Functional studies on human Pex7p: subcellular localization and interaction with proteins containing a peroxisome-targeting signal type 2 and other peroxins.
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ABSTRACT:
SUBMITTER: Ghys K
PROVIDER: S-EPMC1222642 | biostudies-other | 2002 Jul
REPOSITORIES: biostudies-other
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Project description:AcCATPO is a plant catalase-phenol oxidase recently identified from red amaranth. Its physiological function remains unexplored. As the starting step of functional analysis, here we report its subcellular localization and a non-canonical targeting signal. Commonly used bioinformatics programs predicted a peroxisomal localization for AcCATPO, but failed in identification of canonical peroxisomal targeting signals (PTS). The C-terminal GFP tagging led the fusion protein AcCATPO-GFP to the cytosol and the nucleus, but N-terminal tagging directed the GFP-AcCATPO to peroxisomes and nuclei, in transgenic tobacco. Deleting the tripeptide (PTM) at the extreme C-terminus almost ruled out the peroxisomal localization of GFP-AcCATPO?3, and removing the C-terminal decapeptide completely excluded peroxisomes as the residence of GFP-AcCATPO?10. Furthermore, this decapeptide as a targeting signal could import GFP-10aa to the peroxisome exclusively. Taken together, these results demonstrate that AcCATPO is localized to the peroxisome and the nucleus, and its peroxisomal localization is attributed to a non-canonical PTS1, the C-terminal decapeptide which contains an internal SRL motif and a conserved tripeptide P-S/T-I/M at the extreme of C-terminus. This work may further the study as to the physiological function of AcCATPO, especially clarify its involvement in betalain biosynthesis, and provide a clue to elucidate more non-canonic PTS.
| S-EPMC5539789 | biostudies-literature
Project description:BackgroundProteins that interact in vivo tend to reside within the same or "adjacent" subcellular compartments. This observation provides opportunities to reveal protein subcellular localization in the context of the protein-protein interaction (PPI) network. However, so far, only a few efforts based on heuristic rules have been made in this regard.ResultsWe systematically and quantitatively validate the hypothesis that proteins physically interacting with each other probably share at least one common subcellular localization. With the result, for the first time, four graph-based semi-supervised learning algorithms, Majority, χ2-score, GenMultiCut and FunFlow originally proposed for protein function prediction, are introduced to assign "multiplex localization" to proteins. We analyze these approaches by performing a large-scale cross validation on a Saccharomyces cerevisiae proteome compiled from BioGRID and comparing their predictions for 22 protein subcellular localizations. Furthermore, we build an ensemble classifier to associate 529 unlabeled and 137 ambiguously-annotated proteins with subcellular localizations, most of which have been verified in the previous experimental studies.ConclusionsPhysical interaction of proteins has actually provided an essential clue for their co-localization. Compared to the local approaches, the global algorithms consistently achieve a superior performance.
| S-EPMC3314587 | biostudies-literature
Project description:BackgroundThe import of most intraperoxisomal proteins is mediated by peroxisome targeting signals at their C-termini (PTS1) or N-terminal regions (PTS2). Both signals have been integrated in subcellular location prediction programs. However their present performance, particularly of PTS2-targeting did not seem fitting for large-scale screening of sequences.ResultsWe modified an earlier reported PTS1 screening method to identify PTS2-containing mouse candidates using a combination of computational and manual annotation. For rapid confirmation of five new PTS2- and two previously identified PTS1-containing candidates we developed the new cell line CHO-perRed which stably expresses the peroxisomal marker dsRed-PTS1. Using CHO-perRed we confirmed the peroxisomal localization of PTS1-targeted candidate Zadh2. Preliminary characterization of Zadh2 expression suggested non-PPARalpha mediated activation. Notably, none of the PTS2 candidates located to peroxisomes.ConclusionIn a few cases the PTS may oscillate from "silent" to "functional" depending on its surface accessibility indicating the potential for context-dependent conditional subcellular sorting. Overall, PTS2-targeting predictions are unlikely to improve without generation and integration of new experimental data from location proteomics, protein structures and quantitative Pex7 PTS2 peptide binding assays.
| S-EPMC2638156 | biostudies-literature
Project description:Four hundred and fifty-eight genes coding for PentatricoPeptide Repeat (PPR) proteins are annotated in the Arabidopsis thaliana genome. Over the past 10 years, numerous reports have shown that many of these proteins function in organelles to target specific transcripts and are involved in post-transcriptional regulation. Therefore, they are thought to be important players in the coordination between nuclear and organelle genome expression. Only four of these proteins have been described to be addressed outside organelles, indicating that some PPRs could function in post-transcriptional regulations of nuclear genes. In this work, we updated and improved our current knowledge on the localization of PPR proteins of Arabidopsis within the plant cell. We particularly investigated the subcellular localization of 166 PPR proteins whose targeting predictions were ambiguous, using a combination of high-throughput cloning and microscopy. Through systematic localization experiments and data integration, we confirmed that PPR proteins are largely targeted to organelles and showed that dual targeting to both the mitochondria and plastid occurs more frequently than expected. These results allow us to speculate that dual-targeted PPR proteins could be important for the fine coordination of gene expressions in both organelles.
| S-EPMC3858439 | biostudies-literature
Project description:Based on peroxin protein 5 (Pex5p) homology searches in the expressed sequence tag database and sequencing of large full-length cDNA inserts, three novel and related human cDNAs were identified. The brain-derived cDNAs coded for two related proteins that differ only slightly at their N-terminus, and exhibit 39.8% identity to human PEX5p. The shorter liver-derived cDNA coded for the C-terminal tetratricopeptide repeat-containing domain of the brain cDNA-encoded proteins. Since these three proteins specifically bind to various C-terminal peroxisome-targeting signals in a manner indistinguishable from Pex5p and effectively compete with Pex5p in an in vitro peroxisome-targeting signal 1 (PTS1)-binding assay, we refer to them as 'Pex5p-related proteins' (Pex5Rp). In contrast to Pex5p, however, human PEX5Rp did not bind to Pex14p or to the RING finger motif of Pex12p, and could not restore PTS1 protein import in Pex5(-/-) mouse fibroblasts. Immunofluorescence analysis of epitope-tagged PEX5Rp in Chinese hamster ovary cells suggested an exclusively cytosolic localization. Northern-blot analysis showed that the PEX5R gene, which is localized to chromosome 3q26.2--3q27, is expressed preferentially in brain. Mouse PEX5Rp was also delineated. In addition, experimental evidence established that the closest-related yeast homologue, YMR018wp, did not bind PTS1. Based on its subcellular localization and binding properties, Pex5Rp may function as a regulator in an early step of the PTS1 protein import process.
| S-EPMC1221994 | biostudies-other
Project description:BackgroundThe computational prediction of mycobacterial proteins' subcellular localization is of key importance for proteome annotation and for the identification of new drug targets and vaccine candidates. Several subcellular localization classifiers have been developed over the past few years, which have comprised both general localization and feature-based classifiers. Here, we have validated the ability of different bioinformatics approaches, through the use of SignalP 2.0, TatP 1.0, LipoP 1.0, Phobius, PA-SUB 2.5, PSORTb v.2.0.4 and Gpos-PLoc, to predict secreted bacterial proteins. These computational tools were compared in terms of sensitivity, specificity and Matthew's correlation coefficient (MCC) using a set of mycobacterial proteins having less than 40% identity, none of which are included in the training data sets of the validated tools and whose subcellular localization have been experimentally confirmed. These proteins belong to the TBpred training data set, a computational tool specifically designed to predict mycobacterial proteins.ResultsA final validation set of 272 mycobacterial proteins was obtained from the initial set of 852 mycobacterial proteins. According to the results of the validation metrics, all tools presented specificity above 0.90, while dispersion sensitivity and MCC values were above 0.22. PA-SUB 2.5 presented the highest values; however, these results might be biased due to the methodology used by this tool. PSORTb v.2.0.4 left 56 proteins out of the classification, while Gpos-PLoc left just one protein out.ConclusionBoth subcellular localization approaches had high predictive specificity and high recognition of true negatives for the tested data set. Among those tools whose predictions are not based on homology searches against SWISS-PROT, Gpos-PLoc was the general localization tool with the best predictive performance, while SignalP 2.0 was the best tool among the ones using a feature-based approach. Even though PA-SUB 2.5 presented the highest metrics, it should be taken into account that this tool was trained using all proteins reported in SWISS-PROT, which includes the protein set tested in this study, either as a BLAST search or as a training model.
| S-EPMC2685389 | biostudies-literature
Project description:The peroxisome targeting signal type 1 (PTS1) receptor, Pex5p, of the tetratricopeptide repeat (TPR) motif family is located mostly in the cytosol and mediates the translocation of PTS1 proteins to peroxisomes. As a step towards understanding the mechanisms of protein import into peroxisomes, we investigated the molecular mechanisms involved in PTS1 recognition by Pex5p with regard to requirement of energy and cytosolic factors, using cell-free synthesized acyl-CoA oxidase (AOx) as a PTS1 cargo protein, together with Pex5p and heat-shock protein (Hsp)70 from rat liver. Pex5p was partly associated with peroxisomes of rat liver, was resistant to washing with a high concentration of salt and to alkaline extraction and was inaccessible to protease added externally. Pex5p bound to AOx in an ATP-dependent manner. AOx synthesized in a cell-free translating system from rabbit reticulocyte lysate was imported into peroxisomes without being supplemented with Pex5p and Hsp70, implying that peroxisome-associated Pex5p was released from the membranes and functional in this in vitro import assay. Antibodies against Pex5p and Hsp70 inhibited AOx import. In contrast, AOx synthesized in a wheat-germ lysate required the external addition of Pex5p for import, in which Hsp70 augmented the AOx import. The TPR domain of Pex5p was revealed to bind to the N-terminal part in an Hsp70-independent manner, whereas mutual interaction of the TPR region was noted in the presence of Hsp70. Hsp70 interacted with the TPR domain of Pex5p. Moreover, Hsp70 and ATP synergistically enhanced the binding of Pex5p to the C-terminal PTS1-containing part of AOx, implying that Pex5p recognizes its cargo PTS1 protein by chaperone-assisted as well as energy-dependent mechanisms in vivo.
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Project description:Fanconi anemia (FA) is a rare recessive disease, characterized by congenital defects, bone marrow failure, and increased cancer susceptibility. FA is caused by biallelic mutation of any one of sixteen genes. The protein products of these genes function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). A central step in the activation of this pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Monoubiquitinated FANCD2 and FANCI localize to discrete chromatin regions where they function in ICL repair. Despite their critical role in ICL repair, very little is known about the structure, function, and regulation of the FANCD2 and FANCI proteins, or how they are targeted to the nucleus and chromatin. In this study, we describe the functional characterization of an amino-terminal FANCD2 nuclear localization signal (NLS). We demonstrate that the amino terminal 58 amino acids of FANCD2 can promote the nuclear expression of GFP and is necessary for the nuclear localization of FANCD2. Importantly, mutation of this FANCD2 NLS reveals that intact FANCD2 is required for the nuclear localization of a subset of FANCI. In addition, the NLS is necessary for the efficient monoubiquitination of FANCD2 and FANCI and, consequently, for their localization to chromatin. As a result, FANCD2 NLS mutants fail to rescue the ICL sensitivity of FA-D2 patient cells. Our studies yield important insight into the domain structure of the poorly characterized FANCD2 protein, and reveal a previously unknown mechanism for the coordinate nuclear import of a subset of FANCD2 and FANCI, a key early step in the cellular ICL response.
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Project description:BackgroundThe expansion of raw protein sequence databases in the post genomic era and availability of fresh annotated sequences for major localizations particularly motivated us to introduce a new improved version of our previously forged eukaryotic subcellular localizations prediction method namely "ESLpred". Since, subcellular localization of a protein offers essential clues about its functioning, hence, availability of localization predictor would definitely aid and expedite the protein deciphering studies. However, robustness of a predictor is highly dependent on the superiority of dataset and extracted protein attributes; hence, it becomes imperative to improve the performance of presently available method using latest dataset and crucial input features.ResultsHere, we describe augmentation in the prediction performance obtained for our most popular ESLpred method using new crucial features as an input to Support Vector Machine (SVM). In addition, recently available, highly non-redundant dataset encompassing three kingdoms specific protein sequence sets; 1198 fungi sequences, 2597 from animal and 491 plant sequences were also included in the present study. First, using the evolutionary information in the form of profile composition along with whole and N-terminal sequence composition as an input feature vector of 440 dimensions, overall accuracies of 72.7, 75.8 and 74.5% were achieved respectively after five-fold cross-validation. Further, enhancement in performance was observed when similarity search based results were coupled with whole and N-terminal sequence composition along with profile composition by yielding overall accuracies of 75.9, 80.8, 76.6% respectively; best accuracies reported till date on the same datasets.ConclusionThese results provide confidence about the reliability and accurate prediction of SVM modules generated in the present study using sequence and profile compositions along with similarity search based results. The presently developed modules are implemented as web server "ESLpred2" available at http://www.imtech.res.in/raghava/eslpred2/.
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Project description:Hantavirus assembly and budding are governed by the surface glycoproteins Gn and Gc. In this study, we investigated the glycoproteins of Puumala, the most abundant Hantavirus species in Europe, using fluorescently labeled wild-type constructs and cytoplasmic tail (CT) mutants. We analyzed their intracellular distribution, co-localization and oligomerization, applying comprehensive live, single-cell fluorescence techniques, including confocal microscopy, imaging flow cytometry, anisotropy imaging and Number&Brightness analysis. We demonstrate that Gc is significantly enriched in the Golgi apparatus in absence of other viral components, while Gn is mainly restricted to the endoplasmic reticulum (ER). Importantly, upon co-expression both glycoproteins were found in the Golgi apparatus. Furthermore, we show that an intact CT of Gc is necessary for efficient Golgi localization, while the CT of Gn influences protein stability. Finally, we found that Gn assembles into higher-order homo-oligomers, mainly dimers and tetramers, in the ER while Gc was present as mixture of monomers and dimers within the Golgi apparatus. Our findings suggest that PUUV Gc is the driving factor of the targeting of Gc and Gn to the Golgi region, while Gn possesses a significantly stronger self-association potential.
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