OutCyte: a novel tool for predicting unconventional protein secretion

Abstract

The prediction of protein localization, such as in the extracellular space, from high-throughput data is essential for functional downstream inference. It is well accepted that some secreted proteins go through the classic endoplasmic reticulum-Golgi pathway with the guidance of a signal peptide. However, a large number of proteins have been found to reach the extracellular space by following unconventional secretory pathways. There remains a demand for reliable prediction of unconventional protein secretion (UPS). Here, we present OutCyte, a fast and accurate tool for the prediction of UPS, which for the first time has been built upon experimentally determined UPS proteins. OutCyte mediates the prediction of protein secretion in two steps: first, proteins with N-terminal signals are accurately filtered out; second, proteins without N-terminal signals are classified as UPS or intracellular proteins based on physicochemical features directly generated from their amino acid sequences. We are convinced that OutCyte will play a relevant role in the annotation of experimental data and will therefore contribute to further characterization of the extracellular nature of proteins by considering the commonly neglected UPS proteins.OutCyte has been implemented as a web server at www.outcyte.com.

Comparative Secretomics Gives Access to High Confident Secretome Data: Evaluation of Different Methods for the Determination of Bona Fide Secreted Proteins

“…One example from the A549 dataset is the known UPS protein HMGB1 which has been shown to be misclassified by OutCyte. [ 7 ] Further examples are well‐known proteins with extracellular function like disintegrin and metalloproteinase domain‐containing protein 10, High mobility group protein B2, Sulfhydryl oxidase 1, and Vascular cell adhesion protein 1 (Data S3, Supporting Information). Noteworthy, 38% (A549), 91% (MSC), and 49% (NHDF) of those unassigned proteins exhibit an annotated transmembrane domain, and hence are candidates for ectodomain shedding.…”

“…In this context, the predictive power of pure bioinformatic approaches should not be overestimated. Although the fact that the prediction of UPS has improved, [ 7 ] the in silico annotation is still error prone. Moreover, we cannot be excluded that also the prediction of signal peptides and therewith the assignment to the classically secretory pathway is partly incorrect as, for example, isoform information could only partly be considered in shotgun approaches.…”

“…HMGB1 and its family member HMGB2 which we have also detected in the secretome of A549 cells are known to be unconventionally secreted [ 30,31 ] but are misclassified by OutCyte. [ 7 ] HMGB1, a non‐histone nuclear factor, acts extracellularly as a mediator of delayed endotoxin lethality and its secretion is induced by stimuli triggering lysosome exocytosis. [ 30 ] We speculate that the physicochemical signature of some unconventionally secreted proteins like the High Mobility Group B proteins are probably underrepresented in currently available secretome data and therefore should be considered in the development of advanced prediction tools in the future.…”

“…The in silico methods rely on database annotations or are so far mainly based on protein sequence information which has been used for the development of prediction tools like SignalP, [ 3 ] Phobius, [ 4 ] SPOCTOPUS, [ 5 ] SecretomeP, [ 6 ] OutCyte, [ 7 ] SecretomeP, or database annotations. [ 8 ] Analyzing data from transcriptome and proteome studies, Uhlen and colleagues predicted that the human “classical” secretome consists of 2641 proteins.…”

Secretome Analysis of Mesenchymal Stem Cell Factors Fostering Oligodendroglial Differentiation of Neural Stem Cells In Vivo

“…To predict unconventionally secreted proteins, we used the OutCyte prediction tool [74], wherein we selected classically secreted proteins on the basis of the annotation from UniProtKB (signal peptide predicted, but neither a transmembrane domain nor a kdel sequence present). Gene Ontology (GO) biological process (GOBP) and cellular compartment (GOCC) were used for categorical annotations of proteins identified in the MSC-derived secretome, and annotation enrichments were calculated by Fisher's exact tests.…”