A Membrane-Assisted Mechanism for the Release of Ceramide from the CERT START Domain.

Ceramide transfer protein CERT is the mediator of nonvesicular transfer of ceramide from the ER to Golgi. In CERT, START is the domain responsible for the binding and transport of ceramide. A wealth of structural data has revealed a helix-grip fold surrounding a large hydrophobic cavity holding the ceramide. Yet, little is known about the mechanisms by which START releases the ceramide through the polar region and into the packed environment of cellular membranes. As such events do not lend themselves easily to experimental investigations, we used multiple unbiased microsecond-long molecular simulations. We propose a membrane-assisted mechanism in which the membrane acts as an allosteric effector initiating the release of ceramide and where the passage of the ceramide acyl chains is facilitated by the intercalation of a single phosphatidylcholine lipid in the cavity, practically greasing the ceramide way out. We verify using free energy calculation and experimental lipidomics data that CERT forms stable complexes with phosphatidylcholine lipids, in addition to ceramide, thus providing validation for the proposed mechanism.

Isoform- and cell-state-specific lipidation of ApoE in astrocytes.

Apolipoprotein E transports lipids and couples metabolism between astrocytes and neurons. The E4 variant (APOE4) affects these functions and represents a genetic predisposition for Alzheimer's disease, but the molecular mechanisms remain elusive. We show that ApoE produces different types of lipoproteins via distinct lipidation pathways. ApoE forms high-density lipoprotein (HDL)-like, cholesterol-rich particles via the ATP-binding cassette transporter 1 (ABCA1), a mechanism largely unaffected by ApoE polymorphism. Alternatively, ectopic accumulation of fat in astrocytes, a stress-associated condition, redirects ApoE toward the assembly and secretion of triacylglycerol-rich lipoproteins, a process boosted by the APOE4 variant. We demonstrate in vitro that ApoE can detect triacylglycerol in membranes and spontaneously assemble lipoprotein particles (10-20 nm) rich in unsaturated triacylglycerol, and that APOE4 has remarkable properties behaving as a strong triacylglycerol binder. We propose that fatty APOE4 astrocytes have reduced ability to clear toxic fatty acids from the extracellular milieu, because APOE4 reroutes them back to secretion.

Discovering cellular protein-protein interactions: Technological strategies and opportunities.

The analysis of protein interaction networks is one of the key challenges in the study of biology. It connects genotypes to phenotypes, and disruption often leads to diseases. Hence, many technologies have been developed to study protein-protein interactions (PPIs) in a cellular context. The expansion of the PPI technology toolbox however complicates the selection of optimal approaches for diverse biological questions. This review gives an overview of the binary and co-complex technologies, with the former evaluating the interaction of two co-expressed genetically tagged proteins, and the latter only needing the expression of a single tagged protein or no tagged proteins at all. Mass spectrometry is crucial for some binary and all co-complex technologies. After the detailed description of the different technologies, the review compares their unique specifications, advantages, disadvantages, and applicability, while highlighting opportunities for further advancements.

Analyzing trapped protein complexes by Virotrap and SFINX.

The analysis of protein interaction networks is one of the key challenges in the study of biology. It connects genotypes to phenotypes, and disruption of such networks is associated with many pathologies. Virtually all the approaches to the study of protein complexes require cell lysis, a dramatic step that obliterates cellular integrity and profoundly affects protein interactions. This protocol starts with Virotrap, a novel approach that avoids the need for cell homogenization by fusing the protein of interest to the HIV-1 Gag protein, trapping protein complexes in virus-like particles. By using the straightforward filtering index (SFINX), which is a powerful and intuitive online tool (http://sfinx.ugent.be) that enables contaminant removal from candidate lists resulting from mass-spectrometry-based analysis, we provide a complete workflow for researchers interested in mammalian protein complexes. Given direct access to mass spectrometers, researchers can process up to 24 samples in 7 d.

sfinx: an R package for the elimination of false positives from affinity purification-mass spectrometry datasets.

SUMMARY: We describe sfinx, an R package providing access to the straightforward filtering index (SFINX) for the separation of true positive from false positive protein interactions in affinity purification - mass spectrometry datasets. This package maintains the reliability and user-friendliness of the SFINX web site interface but is faster, unlimited in input size, and can be run locally within R. AVAILABILITY AND IMPLEMENTATION: The sfinx R package is available for download at the comprehensive R archive network (CRAN) https://cran.r-project.org/web/packages/sfinx/ under the Apache License 2.0. CONTACT: sven.eyckerman@vib-ugent.be or kevin.titeca@gmail.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Protein complex analysis: From raw protein lists to protein interaction networks.

The elucidation of molecular interaction networks is one of the pivotal challenges in the study of biology. Affinity purification-mass spectrometry and other co-complex methods have become widely employed experimental techniques to identify protein complexes. These techniques typically suffer from a high number of false negatives and false positive contaminants due to technical shortcomings and purification biases. To support a diverse range of experimental designs and approaches, a large number of computational methods have been proposed to filter, infer and validate protein interaction networks from experimental pull-down MS data. Nevertheless, this expansion of available methods complicates the selection of the most optimal ones to support systems biology-driven knowledge extraction. In this review, we give an overview of the most commonly used computational methods to process and interpret co-complex results, and we discuss the issues and unsolved problems that still exist within the field. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 36:600-614, 2017.

Trapping mammalian protein complexes in viral particles.

Cell lysis is an inevitable step in classical mass spectrometry-based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes.

SFINX: Straightforward Filtering Index for Affinity Purification-Mass Spectrometry Data Analysis.

Affinity purification-mass spectrometry is one of the most common techniques for the analysis of protein-protein interactions, but inferring bona fide interactions from the resulting data sets remains notoriously difficult. We introduce SFINX, a Straightforward Filtering INdeX that identifies true-positive protein interactions in a fast, user-friendly, and highly accurate way. SFINX outperforms alternative techniques on two benchmark data sets and is available via the Web interface at http://sfinx.ugent.be/.