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 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

Prevalence of hepatitis B virus marker positivity and evolution of hepatitis B virus profile, during chemotherapy, in patients with solid tumours

To prospectively evaluate the prevalence of hepatitis B virus (HBV) positivity and study the evolution of HBV profile during cancer chemotherapy, serum HBV markers and liver biochemistry were determined in 1008 of 1402 (72%) cancer patients admitted in our Unit and in all 920 (91%) who received chemotherapy. We found that 54 (5.3%) were HBsAg carriers while 443 (44%) had at least one HBV marker positive. Of the latter, 405 (91%) were HBcAb+ve, 321 (72%) HBsAb+ve and 212 (48%) HBeAb+ve. No patient was HBeAg+ve. Among 920 chemotherapy receivers, 374 (41%) were HBcAb+ve, 280 (30%) HBsAb+ve and 178 (19%) HBeAb+ve. Fifty (5.4%) were HBsAg carriers (versus 0.6% in Greek blood donors). All 50 were systematically screened for HBsAg and HBsAb status throughout chemotherapy, during follow-up or until their death, and liver biochemistry was performed before each chemotherapy course. Stable antigenaemia was observed in 43/50 (86%) while 7/50 (14%) developed clinical and/or biochemical hepatitis. Six of these seven developed serum anti-HBs antibodies with an associated decrease of serum HBsAg titres. We conclude that reactivation of HBV infection during chemotherapy is not rare (14%), while disappearance of HBs antigenaemia is neither a frequent nor usually a permanent phenomenon. © 1999 Cancer Research Campaig

Intra- and interreader reproducibility of PI-RADSv2: A multireader study

Background: The Prostate Imaging Reporting and Data System version 2 (PI-RADSv2) has been in use since 2015; while interreader reproducibility has been studied, there has been a paucity of studies investigating the intrareader reproducibility of PI-RADSv2. Purpose: To evaluate both intra- and interreader reproducibility of PI-RADSv2 in the assessment of intraprostatic lesions using multiparametric magnetic resonance imaging (mpMRI). Study Type: Retrospective. Population/Subjects: In all, 102 consecutive biopsy-naïve patients who underwent prostate MRI and subsequent MR/transrectal ultrasonography (MR/TRUS)-guided biopsy. Field Strength/Sequences: Prostate mpMRI at 3T using endorectal with phased array surface coils (TW MRI, DW MRI with ADC maps and b2000 DW MRI, DCE MRI). Assessment: Previously detected and biopsied lesions were scored by four readers from four different institutions using PI-RADSv2. Readers scored lesions during two readout rounds with a 4-week washout period. Statistical Tests: Kappa (κ) statistics and specific agreement (Po) were calculated to quantify intra- and interreader reproducibility of PI-RADSv2 scoring. Lesion measurement agreement was calculated using the intraclass correlation coefficient (ICC). Results: Overall intrareader reproducibility was moderate to substantial (κ = 0.43–0.67, Po = 0.60–0.77), while overall interreader reproducibility was poor to moderate (κ = 0.24, Po = 46). Readers with more experience showed greater interreader reproducibility than readers with intermediate experience in the whole prostate (P = 0.026) and peripheral zone (P = 0.002). Sequence-specific interreader agreement for all readers was similar to the overall PI-RADSv2 score, with κ = 0.24, 0.24, and 0.23 and Po = 0.47, 0.44, and 0.54 in T2-weighted, diffusion-weighted imaging (DWI), and dynamic contrast-enhanced (DCE), respectively. Overall intrareader and interreader ICC for lesion measurement was 0.82 and 0.71, respectively. Data Conclusion: PI-RADSv2 provides moderate intrareader reproducibility, poor interreader reproducibility, and moderate interreader lesion measurement reproducibility. These findings suggest a need for more standardized reader training in prostate MRI. Level of Evidence: 2. Technical Efficacy: Stage 2

Impact Monitoring of the National Scale Up of Zinc Treatment for Childhood Diarrhea in Bangladesh: Repeat Ecologic Surveys

Charles Larson and colleagues find that 23 months into a national campaign to scale up zinc treatment for diarrhea in children under age 5 years, only 10% of children with diarrhea in rural areas and 20%–25% in urban/municipal areas were getting the treatment

Caffeic Acid Phenethyl Ester Causes p21Cip1 Induction, Akt Signaling Reduction, and Growth Inhibition in PC-3 Human Prostate Cancer Cells

Caffeic acid phenethyl ester (CAPE) treatment suppressed proliferation, colony formation, and cell cycle progression in PC-3 human prostate cancer cells. CAPE decreased protein expression of cyclin D1, cyclin E, SKP2, c-Myc, Akt1, Akt2, Akt3, total Akt, mTOR, Bcl-2, Rb, as well as phosphorylation of Rb, ERK1/2, Akt, mTOR, GSK3α, GSK3β, PDK1; but increased protein expression of KLF6 and p21Cip1. Microarray analysis indicated that pathways involved in cellular movement, cell death, proliferation, and cell cycle were affected by CAPE. Co-treatment of CAPE with chemotherapeutic drugs vinblastine, paclitaxol, and estramustine indicated synergistic suppression effect. CAPE administration may serve as a potential adjuvant therapy for prostate cancer

Stellar Coronal and Wind Models: Impact on Exoplanets

Surface magnetism is believed to be the main driver of coronal heating and stellar wind acceleration. Coronae are believed to be formed by plasma confined in closed magnetic coronal loops of the stars, with winds mainly originating in open magnetic field line regions. In this Chapter, we review some basic properties of stellar coronae and winds and present some existing models. In the last part of this Chapter, we discuss the effects of coronal winds on exoplanets.Comment: Chapter published in the "Handbook of Exoplanets", Editors in Chief: Juan Antonio Belmonte and Hans Deeg, Section Editor: Nuccio Lanza. Springer Reference Work