Structural requirements for interaction of peroxisomal targeting signal 2 and its receptor PEX7.

The import of a subset of peroxisomal matrix proteins is mediated by the peroxisomal targeting signal 2 (PTS2). The results of our sequence and physical property analysis of known PTS2 signals and of a mutational study of the least characterized amino acids of a canonical PTS2 motif indicate that PTS2 forms an amphipathic helix accumulating all conserved residues on one side. Three-dimensional structural modeling of the PTS2 receptor PEX7 reveals a groove with an evolutionarily conserved charge distribution complementary to PTS2 signals.

pkaPS: prediction of protein kinase A phosphorylation sites with the simplified kinase-substrate binding model.

Background: Protein kinase A (cAMP-dependent kinase, PKA) is a serine/threonine kinase, for which ca. 150 substrate proteins are known. Based on a refinement of the recognition motif using the available experimental data, we wished to apply the simplified substrate protein binding model for accurate prediction of PKA phosphorylation sites, an approach that was previously successful for the prediction of lipid posttranslational modifications and of the PTS1 peroxisomal translocation signal.

Application of a sensitive collection heuristic for very large protein families: evolutionary relationship between adipose triglyceride lipase (ATGL) and classic mammalian lipases.

Background: Manually finding subtle yet statistically significant links to distantly related homologues becomes practically impossible for very populated protein families due to the sheer number of similarity searches to be invoked and analyzed. The unclear evolutionary relationship between classical mammalian lipases and the recently discovered human adipose triglyceride lipase (ATGL; a patatin family member) is an exemplary case for such a problem.

Results: We describe an unsupervised, sensitive sequence segment collection heuristic suitable for assembling very large protein families.

Hidden localization motifs: naturally occurring peroxisomal targeting signals in non-peroxisomal proteins.

Background: Can sequence segments coding for subcellular targeting or for posttranslational modifications occur in proteins that are not substrates in either of these processes? Although considerable effort has been invested in achieving low false-positive prediction rates, even accurate sequence-analysis tools for the recognition of these motifs generate a small but noticeable number of protein hits that lack the appropriate biological context but cannot be rationalized as false positives.

Results: We show that the carboxyl termini of a set of definitely non-peroxisomal proteins with predicted peroxisomal targeting signals interact with the peroxisomal matrix protein receptor peroxin 5 (PEX5) in a yeast two-hybrid test. Moreover, we show that examples of these proteins - chicken lysozyme, human tyrosinase and the yeast mitochondrial ribosomal protein L2 (encoded by MRP7) - are imported into peroxisomes in vivo if their original sorting signals are disguised.

Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase.

Mobilization of fatty acids from triglyceride stores in adipose tissue requires lipolytic enzymes. Dysfunctional lipolysis affects energy homeostasis and may contribute to the pathogenesis of obesity and insulin resistance. Until now, hormone-sensitive lipase (HSL) was the only enzyme known to hydrolyze triglycerides in mammalian adipose tissue.

Prediction of sequence signals for lipid post-translational modifications: insights from case studies.

In silico annotation techniques for post-translational modifications (PTMs) are important to generate biologically meaningful descriptions for sequences of experimentally uncharacterized proteins. Having previously contributed with predictors for lipid PTMs, we summarize our methodological experience. Rather than only looking for the sequence pattern in substrate sequences, a strategy aimed at creating a generalized model of substrate protein/enzyme interaction appears more appropriate since the number of known substrate sequences is small, and some of them are not sufficiently verified experimentally.

Prediction of lipid posttranslational modifications and localization signals from protein sequences: big-Pi, NMT and PTS1.

Many posttranslational modifications (N-myristoylation or glycosylphosphatidylinositol (GPI) lipid anchoring) and localization signals (the peroxisomal targeting signal PTS1) are encoded in short, partly compositionally biased regions at the N- or C-terminus of the protein sequence. These sequence signals are not well defined in terms of amino acid type preferences but they have significant interpositional correlations. Although the number of verified protein examples is small, the quantification of several physical conditions necessary for productive protein binding with the enzyme complexes executing the respective transformations can lead to predictors that recognize the signals from the amino acid sequence of queries alone.

Prediction of peroxisomal targeting signal 1 containing proteins from amino acid sequence.

Peroxisomal matrix proteins have to be imported into their target organelle post-translationally. The major translocation pathway depends on a C-terminal targeting signal, termed PTS1. Our previous analysis of sequence variability in the PTS1 motif revealed that, in addition to the known C-terminal tripeptide, at least nine residues directly upstream are important for signal recognition in the PTS1-Pex5 receptor complex.

Motif refinement of the peroxisomal targeting signal 1 and evaluation of taxon-specific differences.

Eukaryote peroxisomes, plant glyoxysomes and trypanosomal glycosomes belong to the microbody family of organelles that compartmentalise a variety of biochemical processes. The interaction between the PTS1 signal and its cognate receptor Pex5 initiates the major import mechanism for proteins into the matrix of these organelles. Relying on the analysis of amino acid sequence variability of known PTS1-targeted proteins and PTS1-containing peptides that interact with Pex5 in the yeast two-hybrid assay, on binding site studies of the Pex5-ligand complex crystal structure, 3D models and sequences of Pex5 proteins from various taxa, we derived the requirements for a C-terminal amino acid sequence to interact productively with Pex5.