Defining hereditary alpha-tryptasemia as a risk/modifying factor for anaphylaxis: are we there yet?

Hereditary a-tryptasemia (HaT) is a common autosomal dominant genet-ic trait with variable penetrance associated with increased serum baseline tryptase (SBT) levels. Clinical manifestations may range from an absence of symptoms to overtly severe and recurrent anaphylaxis. Symptoms have been claimed to result from excessive activation of EGF-like module -con-taining mucin-like hormone receptor-like 2 (EMR2) and protease activat-ed receptor 2 (PAR-2) receptors by a/13-tryptase heterotetramers. Herein, we aimed to review the evidence on whether HaT can be considered a hereditary risk factor or a modifying factor for anaphylaxis. Increased SBT levels have been linked to an increased risk of anaphylaxis. Likewise, recent studies have shown that HaT might be associated with a higher risk of developing anaphylaxis and more severe anaphylaxis. The same has also been shown for patients with clonal mast cell disorders, in whom the co-existence of HaT might lead to a greater propensity for se-vere, potentially life-threatening anaphylaxis. However, studies leading to such conclusions are generally limited in sample size, while other studies have shown opposing results. As such, further studies investigating the po-tential association of HaT with anaphylaxis caused by different triggers, and different severity grades, in both patients with clonal mast cell activa-tion syndromes and the general population are still needed

A mechanistic perspective on pex1 and pex6, two aaa+ proteins of the peroxisomal protein import machinery

In contrast to many protein translocases that use ATP or GTP hydrolysis as the driving force to transport proteins across biological membranes, the peroxisomal matrix protein import machinery relies on a regulated self-assembly mechanism for this purpose and uses ATP hydrolysis only to reset its components. The ATP-dependent protein complex in charge of resetting this machinery—the Receptor Export Module (REM)—comprises two members of the “ATPases Associated with diverse cellular Activities” (AAA+) family, PEX1 and PEX6, and a membrane protein that anchors the ATPases to the organelle membrane. In recent years, a large amount of data on the structure/function of the REM complex has become available. Here, we discuss the main findings and their mechanistic implications.This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project PTDC/BEX-BCM/2311/2014 (POCI-01-0145-FEDER-016613) and the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274). This work is a result of the project NORTE-01-0145-FEDER-000008—Porto Neurosciences and Neurologic Disease Research Initiative at I3S, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). A.B.-B., A.G.P., M.J.F., T.F. and T.A.R. are supported by Fundação para a Ciência e Tecnologia, Programa Operacional Potencial Humano do QREN, and Fundo Social Europeu

Protein transport into peroxisomes: Knowns and unknowns

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and rapidly transported into the organelle by a complex machinery. The data gathered in recent years suggest that this machinery operates through a syringe-like mechanism, in which the shuttling receptor PEX5 - the “plunger” - pushes a newly synthesized protein all the way through a peroxisomal transmembrane protein complex - the “barrel” - into the matrix of the organelle. Notably, insertion of cargo-loaded receptor into the “barrel” is an ATP-independent process, whereas extraction of the receptor back into the cytosol requires its monoubiquitination and the action of ATP-dependent mechanoenzymes. Here, we review the main data behind this model.We would like to thank Dr. Marc Fransen (KU Leuven) for his critical reading of the manuscript. This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional, funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministerio da Ciência, Tecnologia e Inovação in the framework of the projects “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) and “Themolecular mechanisms of peroxisome biogenesis” (PTDC/BEX-BCM/2311/2014), and through 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).” T.F., T.A.R., A.F.D., A.B.B., and D.B. were supported by Fundação para a Ciência e a Tecnologia, Programa Operacional Potencial Humano do QREN and Fundo Social Europeu

The peroxisomal matrix protein translocon is a large cavity-forming protein assembly into which PEX5 protein enters to release its cargo

A remarkable property of the machinery for import of peroxisomal matrix proteins is that it can accept already folded proteins as substrates. This import involves binding of newly synthesized proteins by cytosolic peroxisomal biogenesis factor 5 (PEX5) followed by insertion of the PEX5– cargo complex into the peroxisomal membrane at the docking/translocation module (DTM). However, how these processes occur remains largely unknown. Here, we used truncated PEX5 molecules to probe the DTM architecture. We found that the DTM can accommodate a larger number of truncated PEX5 molecules comprising amino acid residues 1–197 than full-length PEX5 molecules. A shorter PEX5 version (PEX5(1–125)) still interacted correctly with the DTM; however, this species was largely accessible to exoge-nously added proteinase K, suggesting that this protease can access the DTM occupied by a small PEX5 protein. Interestingly, the PEX5(1–125)–DTM interaction was inhibited by a polypeptide comprising PEX5 residues 138 – 639. Apparently, the DTM can recruit soluble PEX5 through interactions with different PEX5 domains, suggesting that the PEX5–DTM interactions are to some degree fuzzy. Finally, we found that the interaction between PEX5 and PEX14, a major DTM component, is stable at pH 11.5. Thus, there is no reason to assume that the hitherto intriguing resistance of DTM-bound PEX5 to alkaline extraction reflects its direct contact with the peroxisomal lipid bilayer. Collectively, these results suggest that the DTM is best described as a large cavity-forming protein assembly into which cytosolic PEX5 can enter to release its cargo.This work was supported in part by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020-Operational Pro-gramme for Competitiveness and Internationalization (POCI), Portugal 2020 and by Portuguese funds through Fundação para a Ciência e a Tec-nologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the projects “Institute for Research and Innovation in Health Sciences” (Grant POCI-01-0145-FEDER-007274) and “The molecular mechanisms of peroxisome biogenesis” (Grant PTDC/BEX-BCM/2311/2014) and through Norte 2020-Programa Operacional Regional do Norte under the application of the “Porto Neurosciences and Neurologic Disease Research Initiative at i3S” (Grant NORTE-01-0145-FEDER-000008). The authors declare that they have no conflicts of interest with the contents of this article

Peroxisomal monoubiquitinated PEX5 interacts with the AAA ATPases PEX1 and PEX6 and is unfolded during its dislocation into the cytosol

PEX1 and PEX6 are two members of the ATPases associated with diverse cellular activities (AAA) family and the core components of the receptor export module of the peroxisomal matrix protein import machinery. Their role is to extract monoubiquitinated PEX5, the peroxisomal protein-shuttling receptor, from the peroxisomal membrane docking/translocation module (DTM), so that a new cycle of protein transportation can start. Recent data have shown that PEX1 and PEX6 form a heterohexameric complex that unfolds substrates by processive threading. However, whether the natural substrate of the PEX1-PEX6 complex is monoubiquitinated PEX5 (Ub-PEX5) itself or some Ub-PEX5-interacting component(s) of the DTM remains unknown. In this work, we used an established cell-free in vitro system coupled with photoaffinity cross-linking and protein PEGylation assays to address this problem. We provide evidence suggesting that DTM-embedded Ub-PEX5 interacts directly with both PEX1 and PEX6 through its ubiquitin moiety and that the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event. These findings strongly suggest that DTM-embedded Ub-PEX5 is a bona fide substrate of the PEX1-PEX6 complex.The authors thank Britta Moellers (Ruhr-Universität Bochum, Germany) for providing plasmids and recombinant protein for the generation of the anti-PEX6 antibody. This work was funded by FEDER (Fundo Europeu de Desenvolvimento Regional), through COMPETE 2020 –Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through Fundação para a Ciência e Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Inovação in the framework of the projects “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) and “The molecular mechanisms of peroxisome biogenesis” (PTDC/BEX-BCM/2311/2014), and through 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). A.G.P, T.F., D.B., A.F.D., A.B.B. and T.A.R. are supported by Fundação para a Ciência e Tecnologia, Programa Operacional Potencial Humano do QREN and Fundo Social Europeu

Towards continuously programmable networks

While programmability has been a feature of network devices for a long time, the past decade has seen significant enhancement of programming capability for network functions and nodes, spearheaded by the ongoing trend towards softwarization and cloudification. In his context, new design principles and technology enablers are introduced (Section 7.2) which reside at: (i) service/application provisioning level, (ii) network and resource management level, as well as (iii) network deployment and connectivity level

Fast 3D shape screening of large chemical databases through alignment-recycling

<p>Abstract</p> <p>Background</p> <p>Large chemical databases require fast, efficient, and simple ways of looking for similar structures. Although such tasks are now fairly well resolved for graph-based similarity queries, they remain an issue for 3D approaches, particularly for those based on 3D shape overlays. Inspired by a recent technique developed to compare molecular shapes, we designed a hybrid methodology, alignment-recycling, that enables efficient retrieval and alignment of structures with similar 3D shapes.</p> <p>Results</p> <p>Using a dataset of more than one million PubChem compounds of limited size (< 28 heavy atoms) and flexibility (< 6 rotatable bonds), we obtained a set of a few thousand diverse structures covering entirely the 3D shape space of the conformers of the dataset. Transformation matrices gathered from the overlays between these diverse structures and the 3D conformer dataset allowed us to drastically (100-fold) reduce the CPU time required for shape overlay. The alignment-recycling heuristic produces results consistent with <it>de novo </it>alignment calculation, with better than 80% hit list overlap on average.</p> <p>Conclusion</p> <p>Overlay-based 3D methods are computationally demanding when searching large databases. Alignment-recycling reduces the CPU time to perform shape similarity searches by breaking the alignment problem into three steps: selection of diverse shapes to describe the database shape-space; overlay of the database conformers to the diverse shapes; and non-optimized overlay of query and database conformers using common reference shapes. The precomputation, required by the first two steps, is a significant cost of the method; however, once performed, querying is two orders of magnitude faster. Extensions and variations of this methodology, for example, to handle more flexible and larger small-molecules are discussed.</p