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)

Sex differences in presenting symptoms of acute coronary syndrome: the EPIHeart cohort study

Objectives Prompt diagnosis of acute coronary syndrome (ACS) remains a challenge, with presenting symptoms affecting the diagnosis algorithm and, consequently, management and outcomes. This study aimed to identify sex differences in presenting symptoms of ACS. Design Data were collected within a prospective cohort study (EPIHeart). Setting Patients with confirmed diagnosis of type 1 (primary spontaneous) ACS who were consecutively admitted to the Cardiology Department of two tertiary hospitals in Portugal between August 2013 and December 2014. Participants Presenting symptoms of 873 patients (227 women) were obtained through a face-to-face interview. Outcome measures: Typical pain was defined according to the definition of cardiology societies. Clusters of symptoms other than pain were identified by latent class analysis. Logistic regression was used to quantify differences in presentation of ACS symptoms by sex. Results Chest pain was reported by 82% of patients, with no differences in frequency or location between sexes. Women were more likely to feel pain with an intensity higher than 8/10 and this association was stronger for patients aged under 65 years (interaction P=0.028). Referred pain was also more likely in women, particularly pain referred to typical and atypical locations simultaneously. The multiple symptoms cluster, which was characterised by a high probability of presenting with all symptoms, was almost fourfold more prevalent in women (3.92, 95% CI 2.21 to 6.98). Presentation with this cluster was associated with a higher 30-day mortality rate adjusted for the GRACE V.2.0 risk score (4.9% vs 0.9% for the two other clusters, P<0.001). Conclusions While there are no significant differences in the frequency or location of pain between sexes, women are more likely to feel pain of higher intensity and to present with referred pain and symptoms other than pain. Knowledge of these ACS presentation profiles is important for health policy decisions and clinical practice.This study was funded by FEDER through the Operational Programme Competitiveness and Internationalization and national funding from the Foundation for Science and Technology (FCT; Portuguese Ministry of Science, Technology and Higher Education) (FCOMP-01-0124-FEDER-028709), under the project ’Inequalities in coronary heart disease management and outcomes in Portugal' (Ref. FCT PTDC/DTP-EPI/0434/2012) and Unidade de Investigação em Epidemiologia—Instituto de Saúde Pública da Universidade do Porto (EPIUnit) (POCI-01-0145-FEDER-006862; Ref. UID/DTP/04750/2013)

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)

Inequalities in access to cardiac rehabilitation after an acute coronary syndrome: the EPiHeart cohort

Objectives To estimate cardiac rehabilitation (CR) referral and participation rates among patients with acute coronary syndrome (ACS) and to identify their determinants, in two Portuguese regions. Design Prospective cohort study. Setting Patients consecutively admitted to the cardiology department of two hospitals, one in the district of Porto and one in the north-east region (NER) of Portugal, were enrolled in the EPIHeart cohort and then followed up for 6 months. Participants Between August 2013 and December 2014, 939 patients were included in the cohort, and 853 were re-evaluated at 6-month follow-up. Outcome measures Referral rate was defined as the proportion of eligible patients who were referred to a CR programme, whereas participation rate was defined as the proportion of eligible patients who completed a CR programme, as was recommended by their physicians. Results Patients referred were 32.3% and 10.7% of those eligible in Porto and NER, respectively. In both regions, referral to CR decreased with age and with longer travel times to CR centres and increased with education or social class. At follow-up, 128 patients from Porto (26.2% of those eligible and 81.0% of those referred) and 26 from NER (7.1% of those eligible and 66.7% of those referred) reported actually participating in a CR programme. In Porto, the main barriers to participation were the long time until a programme was available and lack of perceived benefit. Patients in NER identified distance to CR and costs as the main barriers. Conclusions CR remains clearly underused in Portugal, with major inequalities in access between regions. Achieving equitable and greater use of CR requires a multilevel approach addressing barriers related to healthcare system, providers and patients in order to improve provision, referral and participation.This study was supported by FEDER through the Operational Programme Competitiveness and Internationalisation and national funding from the Foundation for Science and Technology—FCT (Portuguese Ministry of Science, Technology and Higher Education) (FCOMP-01-0124-FEDER-028709), under the project ‘Inequalities in coronary heart disease management and outcomes in Portugal’ (FCT PTDC/DTP-EPI/0434/2012) and the Unidade de Investigação em Epidemiologia—Instituto de Saúde Pública da Universidade do Porto (EPIUnit) (POCI-01-0145-FEDER-006862; ref UID/DTP/04750/2013)

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

Water-induced modulation of Helicobacter pylori virulence properties

While the influence of water in Helicobacter pylori culturability and membrane integrity has been extensively studied, there are little data concerning the effect of this environment on virulence properties. Therefore, we studied the culturability of water-exposed H. pylori and determined whether there was any relation with the bacterium’s ability to adhere, produce functional components of pathogenicity and induce inflammation and alterations in apoptosis in an experimental model of human gastric epithelial cells. H. pylori partially retained the ability to adhere to epithelial cells even after complete loss of culturability. However, the microorganism is no longer effective in eliciting in vitro host cell inflammation and apoptosis, possibly due to the non-functionality of the cag type IV secretion system. These H. pylori-induced host cell responses, which are lost along with culturability, are known to increase epithelial cell turnover and, consequently, could have a deleterious effect on the initial H. pylori colonisation process. The fact that adhesion is maintained by H. pylori to the detriment of other factors involved in later infection stages appears to point to a modulation of the physiology of the pathogen after water exposure and might provide the microorganism with the necessary means to, at least transiently, colonise the human stomach.FCT (SFRH/BD/24579/2005) (to NMG

Assessment at Antiretroviral Clinics during TB Treatment Reduces Loss to Follow-Up among HIV-Infected Patients

A South African township clinic where loss to follow-up during TB treatment may prevent HIV-infected TB patients from receiving life-saving ART.To determine factors associated with loss to follow-up during TB treatment.Regression analyses of a cohort of ART-eligible TB patients who commenced TB treatment and were followed for 24 weeks.Of 111 ART-eligible TB patients, 15 (14%) died in the ensuing 24 weeks. Of the remaining 96 TB patients, 11 (11%) were lost to follow-up. All TB patients lost to follow-up did not initiate ART. Of 85 TB patients in follow-up, 62 (73%) initiated ART 56 days after TB diagnosis (median, IQR 33-77 days) and 31 days after initial assessment at an ART clinic (median, IQR: 18-55 days). The median duration from TB diagnosis to initial assessment at an ART clinic was 19 days (IQR: 7-48 days). At 24 weeks, 6 of 85 (7%) TB patients who presented to an ART clinic for assessment were lost to follow-up, compared to 5 of 11 (45%) TB patients who did not present to an ART clinic for assessment. Logistic regression analysis (adjusted odds ratio = 0.1, 95% confidence interval [95% CI]: 0.03-0.66) and our Cox proportional hazards model (hazard ratio = 0.2, 95% CI: 0.04-0.68) confirmed that assessment at an ART clinic during TB treatment reduced loss to follow-up.Assessment at antiretroviral clinics for HIV care by trained health-care providers reduces loss to follow-up among HIV-infected patients with TB