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PubMed2004

Role of reactive oxygen species in ischemic preconditioning of subcellular organelles in the heart.

Ischemic preconditioning (IPC) is an endogenous adaptive mechanism and is manifested by early and delayed phases of cardioprotection. Brief episodes of ischemia-reperfusion during IPC cause some subtle functional and structural alterations in sarcolemma, mitochondria, sarcoplasmic reticulum, myofibrils, glycocalyx, as well as nucleus, which render these subcellular organelles resistant to subsequent sustained ischemia-reperfusion insult. These changes occur in functional groups of various receptors, cation transporters, cation channels, and contractile and other proteins, and may explain the initial effects of IPC. On the other hand, induction of various transcriptional factors occurs to alter gene expression and structural changes in subcellular organelles and may be responsible for the delayed effects of IPC. Reactive oxygen species (ROS), which are formed during the IPC period, may cause these changes directly and indirectly and act as a trigger of IPC-induced cardioprotection. As ROS may be one of the several triggers proposed for IPC, this discussion is focused on the current knowledge of both ROS-dependent and ROS-independent mechanisms of IPC. Furthermore, some events, which are related to functional preservation of subcellular organelles, are described for a better understanding of the IPC phenomenon.

Animals↗

PubMed1997

Subcellular distribution of Xenopus XEL-1 protein, a member of the neuron-specific ELAV/Hu family, revealed by epitope tagging.

Drosophila and vertebrate elav/Hu genes are involved in the development and the maintenance of the nervous system. They all encode proteins that contain three RNA recognition motifs (RRM) and are thus expected to play a role in RNA metabolism. Drosophila ELAV and RBP9 proteins were reported to be exclusively distributed in nuclei of neurons, whereas known human Hu proteins display a bipartite nuclear and cytoplasmic distribution. We have previously isolated a member of this family in Xenopus, Xel-1, that is exclusively expressed in neural tissues from the early tailbud stage onward. In the present study, we report on the subcellular distribution of XEL-1 protein using myc epitope tagging, a strategy allowing the study of a single member of the ELAV/Hu family. We show that the subcellular distribution of exogenous XEL-1 protein in neural tissues depends on developmental stages. In the neural tube at the neurula stage, where endogenous Xel-1 is not expressed, exogenous tagged XEL-1 protein is localized in both the nucleus and the cytoplasm. At the tailbud stage, where endogenous Xel-1 is expressed, exogenous tagged XEL-1 protein is localized essentially in the cytoplasm of neural tube cells. In contrast, exogenous Drosophila ELAV protein localizes to the nucleus at all stages in Xenopus embryos. The variability in the subcellular localization of ELAV/Hu proteins in different species may have functional implications.

Amino Acid Sequence↗

PubMed1996

Different subcellular localization of Saccharomyces cerevisiae HMG-CoA reductase isozymes at elevated levels corresponds to distinct endoplasmic reticulum membrane proliferations.

In all eucaryotic cell types analyzed, proliferations of the endoplasmic reticulum (ER) can be induced by increasing the levels of certain integral ER proteins. One of the best characterized of these proteins is HMG-CoA reductase, which catalyzes the rate-limiting step in sterol biosynthesis. We have investigated the subcellular distributions of the two HMG-CoA reductase isozymes in Saccharomyces cerevisiae and the types of ER proliferations that arise in response to elevated levels of each isozyme. At endogenous expression levels, Hmg1p and Hmg2p were both primarily localized in the nuclear envelope. However, at increased levels, the isozymes displayed distinct subcellular localization patterns in which each isozyme was predominantly localized in a different region of the ER. Specifically, increased levels of Hmg1p were concentrated in the nuclear envelope, whereas increased levels of Hmg2p were concentrated in the peripheral ER. In addition, an Hmg2p chimeric protein containing a 77-amino acid lumenal segment from Hmg1p was localized in a pattern that resembled that of Hmg1p when expressed at increased levels. Reflecting their different subcellular distributions, elevated levels of Hmg1p and Hmg2p induced sets of ER membrane proliferations with distinct morphologies. The ER membrane protein, Sec61p, was localized in the membranes induced by both Hmg1p and Hmg2p green fluorescent protein (GFP) fusions. In contrast, the lumenal ER protein, Kar2p, was present in Hmg1p:GFP membranes, but only rarely in Hmg2p:GFP membranes. These results indicated that the membranes synthesized in response to Hmg1p and Hmg2p were derived from the ER, but that the membranes were not identical in protein composition. We determined that the different types of ER proliferations were not simply due to quantitative differences in protein amounts or to the different half-lives of the two isozymes. It is possible that the specific distributions of the two yeast HMG-CoA reductase isozymes and their corresponding membrane proliferations may reveal regions of the ER that are specialized for certain branches of the sterol biosynthetic pathway.

Endoplasmic Reticulum↗

PubMed2025

Knowledge-enhanced protein subcellular localization prediction from 3D fluorescence microscope images.

MOTIVATION: Pinpointing the subcellular location of proteins is essential for studying protein function and related diseases. Advances in spatial proteomics have shown that automatic recognition of protein subcellular localization from images could highly facilitate protein translocation analysis and biomarker discovery, but existing machine-learning works have been mostly limited to processing 2D images. By contrast, 3D images have higher spatial resolution and allow researchers to observe cellular structures in their natural context, but currently, there are only a few studies of 3D image processing for protein distribution analysis due to the lack of data and complexity of modeling. RESULTS: We developed a knowledge-enhanced protein subcellular localization model, KE3DLoc, which could recognize distribution patterns in 3D fluorescence microscope images using deep learning methods. The model designs an image feature extraction module that incorporates information from 3D and 2D projected cells and implements asymmetric loss and confidence weights to address data imbalance and weak cell annotation issues. Besides, considering that the biological knowledge in the Gene Ontology (GO) database can provide valuable support for protein location understanding, the KE3DLoc model incorporates a novel knowledge enhancement module that optimizes the protein representation by related knowledge graphs derived from the GO. Since the image module and the knowledge module calculate features from different levels, KE3DLoc designs protein ID aggregation to enhance the consistency of protein features across different cells. Experimental results on three public datasets have demonstrated that the KE3DLoc significantly outperforms existing methods and provides valuable insights for spatial proteomics research. AVAILABILITY AND IMPLEMENTATION: All datasets and codes used in this study are available at GitHub: https://github.com/PRBioimages/KE3DLoc.

Microscopy, Fluorescence↗

PubMed2004

Predicting subcellular localization of proteins using machine-learned classifiers.

MOTIVATION: Identifying the destination or localization of proteins is key to understanding their function and facilitating their purification. A number of existing computational prediction methods are based on sequence analysis. However, these methods are limited in scope, accuracy and most particularly breadth of coverage. Rather than using sequence information alone, we have explored the use of database text annotations from homologs and machine learning to substantially improve the prediction of subcellular location. RESULTS: We have constructed five machine-learning classifiers for predicting subcellular localization of proteins from animals, plants, fungi, Gram-negative bacteria and Gram-positive bacteria, which are 81% accurate for fungi and 92-94% accurate for the other four categories. These are the most accurate subcellular predictors across the widest set of organisms ever published. Our predictors are part of the Proteome Analyst web-service.

Algorithms↗

PubMed2006

Quantitative analysis and subcellular distribution of mRNA and protein expression of the hyperpolarization-activated cyclic nucleotide-gated channels throughout development in rat hippocampus.

The properties of the hyperpolarization-activated current (I(h)) and its roles in hippocampal network function evolve radically during development. Because I(h) is conducted by the hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels, we tested the hypothesis that understanding the quantitative developmental profiles of HCN1, HCN2, and HCN4 expression, and the isoform- and age-specific progression of their subcellular distribution, should shed light on the established modifications of the properties of I(h) throughout development. Combined quantitative in situ hybridization, regional western blots, and high-resolution, dual-label immunocytochemistry revealed striking and novel information about the expression and distribution of the HCN channel isoforms in the developing hippocampal formation. In cornus ammon 1 (CA) pyramidal cell layer, a robust increase of HCN1 mRNA and protein expression occurred with age, with reciprocal reduction of HCN4 and relatively stable HCN2 levels. These distinct expression patterns raised the contribution of HCN1 to the total HCN channel pool from 33% to 65% consonant with acceleration and reduced cyclic adenosine mono phosphate (cAMP) sensitivity of I(h) in this region with age. In CA3, strong expression of HCN1 already neonatally supports the recently established role of this conductance in neonatal, age-specific, hippocampal oscillations (giant depolarizing potentials). Notably, HCN1 channels were present and probably transported to dendritic compartments already on postnatal day (P) 2, whereas HCN2 channel protein was not evident in dendrites for the first 2 weeks of life. HCN2 mRNA and protein expression remained fairly constant subsequent to the first week of life in all hippocampal subfields examined, whereas HCN4 mRNA and protein expression declined after maximal neonatal expression, so that the contribution of this isoform to the total HCN channel pool dropped from 43% (CA1) and 34% (CA3) on P11 to 8% (CA1) and 19% (CA3) on P90. Interneuronal expression of all HCN channel isoforms in stratum pyramidale was robust in parvalbumin-but not in cholecystokinin-expressing populations and with a subunit-specific subcellular distribution. Taken together, these data suggest that early in life, HCN4 may contribute significantly to the functions of I(h) in specific hippocampal regions. In addition, these evolving, differential quantitative, and subcellular expression patterns of the HCN channel isoforms support age-specific properties and functions of I(h) within the developing hippocampal formation.

Animals↗

PubMed1998

Using neural networks for prediction of the subcellular location of proteins.

Neural networks have been trained to predict the subcellular location of proteins in prokaryotic or eukaryotic cells from their amino acid composition. For three possible subcellular locations in prokaryotic organisms a prediction accuracy of 81% can be achieved. Assigning a reliability index, 33% of the predictions can be made with an accuracy of 91%. For eukaryotic proteins (excluding plant sequences) an overall prediction accuracy of 66% for four locations was achieved, with 33% of the sequences being predicted with an accuracy of 82% or better. With the subcellular location restricting a protein's possible function, this method should be a useful tool for the systematic analysis of genome data and is available via a server on the world wide web.

Amino Acids↗

PubMed2004

DBSubLoc: database of protein subcellular localization.

We have built a protein subcellular localization annotation database, the DBSubLoc database, which is available at http://www.bioinfo.tsinghua. edu.cn/dbsubloc.html. Annotations were taken from primary protein databases, model organism genome projects and literature texts, and then were analyzed to dig out the subcellular localization features of the proteins. The proteins are also classified into different categories. Based on sequence alignment, non-redundant subsets of the database have been built, which may provide useful information for subcellular localization prediction. The database now contains >60,000 protein sequences including approximately 30,000 protein sequences in the non-redundant data sets. Online download, search and Blast tools are also available.

Animals↗

PubMed2005

LOCSVMPSI: a web server for subcellular localization of eukaryotic proteins using SVM and profile of PSI-BLAST.

Subcellular location of a protein is one of the key functional characters as proteins must be localized correctly at the subcellular level to have normal biological function. In this paper, a novel method named LOCSVMPSI has been introduced, which is based on the support vector machine (SVM) and the position-specific scoring matrix generated from profiles of PSI-BLAST. With a jackknife test on the RH2427 data set, LOCSVMPSI achieved a high overall prediction accuracy of 90.2%, which is higher than the prediction results by SubLoc and ESLpred on this data set. In addition, prediction performance of LOCSVMPSI was evaluated with 5-fold cross validation test on the PK7579 data set and the prediction results were consistently better than the previous method based on several SVMs using composition of both amino acids and amino acid pairs. Further test on the SWISSPROT new-unique data set showed that LOCSVMPSI also performed better than some widely used prediction methods, such as PSORTII, TargetP and LOCnet. All these results indicate that LOCSVMPSI is a powerful tool for the prediction of eukaryotic protein subcellular localization. An online web server (current version is 1.3) based on this method has been developed and is freely available to both academic and commercial users, which can be accessed by at http://Bioinformatics.ustc.edu.cn/LOCSVMPSI/LOCSVMPSI.php.

Artificial Intelligence↗

PubMed2006

LOCATE: a mouse protein subcellular localization database.

We present here LOCATE, a curated, web-accessible database that houses data describing the membrane organization and subcellular localization of proteins from the FANTOM3 Isoform Protein Sequence set. Membrane organization is predicted by the high-throughput, computational pipeline MemO. The subcellular locations of selected proteins from this set were determined by a high-throughput, immunofluorescence-based assay and by manually reviewing >1700 peer-reviewed publications. LOCATE represents the first effort to catalogue the experimentally verified subcellular location and membrane organization of mammalian proteins using a high-throughput approach and provides localization data for approximately 40% of the mouse proteome. It is available at http://locate.imb.uq.edu.au.

Animals↗

PubMed2006

pTARGET: a web server for predicting protein subcellular localization.

The pTARGET web server enables prediction of nine distinct protein subcellular localizations in eukaryotic non-plant species. Predictions are made using a new algorithm [C. Guda and S. Subramaniam (2005) pTARGET [corrected] a new method for predicting protein subcellular localization in eukaryotes. Bioinformatics, 21, 3963-3969], which is primarily based on the occurrence patterns of location-specific protein functional domains in different subcellular locations. We have implemented a relational database, PreCalcDB, to store pre-computed prediction results for all eukaryotic non-plant protein sequences in the public domain that includes about 770,000 entries. Queries can be made by entering protein sequences or by uploading a file containing up to 5000 protein sequences in FASTA format. Prediction results for queries with matching entries in the PreCalcDB will be retrieved instantly; while for the missing ones new predictions will be computed and sent by email. Pre-computed predictions can also be downloaded for complete proteomes of Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila, Mus musculus and Homo sapiens. The server, its documentation and the data are accessible from http://bioinformatics.albany.edu/~ptarget.

Algorithms↗

PubMed2005

Subcellular distribution of Ras GTPase isoforms in normal human kidney.

BACKGROUND: Ras GTPase isoforms have been implicated in proliferative renal disease and are known to have differential cellular expression in kidney. However, their exact subcellular location in various cells is unknown. METHODS: Immunogold labelling for Ras isoforms (Harvey, Kirsten and Neural) was performed for subcellular localization under electron microscopy in fresh normal kidney specimens, obtained from the opposite pole of kidneys removed for renal cell cancer. RESULTS: There was prominent staining shown by Ha-Ras only on the glomerular foot processes as compared with basement membrane or the endothelial cells. Mesangial cells showed intense staining in the cytosol with Ha-Ras (absent in the nucleus), minimal staining with Ki-Ras and none with N-Ras. In both the proximal and distal convoluted tubules, there was a clear staining of the mitochondria with Ha-Ras, with mild cytosolic staining with all of the isoforms. CONCLUSIONS: Ras isoforms have distinct and separate subcellular distributions in normal kidney cells. Understanding the functional aspects of this distribution pattern is essential if Ras is to be targeted by genetic or molecular therapeutic tools.

Humans↗

PubMed1987

Subcellular localization of intracellular forms of human chorionic gonadotropin in first trimester placenta.

To determine the subcellular sites for synthesis and processing of human chorionic gonadotropin subunits in cells, first trimester placental cells were fractionated subcellularly on sucrose density gradients. Analysis of the subcellular fractions by immunobinding techniques revealed that the rough endoplasmic reticulum-rich fraction contained only intermediates having high-mannose oligosaccharides, but the Golgi-rich fraction contained not only intermediates but also mature forms which were resistant to endoglycosidase H but sensitive to neuraminidase. These results show that human chorionic gonadotropin subunits are synthesized in the rough endoplasmic reticulum as forms containing high-mannose oligosaccharides, and their maturation occurs in the Golgi apparatus by trimming with endogenous glycosidases. They are then modified by addition of complex oligosaccharides and terminal sialic acid through glycosyltransferases.

Acetylglucosaminidase↗

PubMed1995

Developmental changes of cyclin dependent kinase 5 subcellular localization in the rat cerebellum.

Cyclin dependent kinase 5 (Cdk5) phosphorylates tau protein, a microtubule-associated protein, at pathological sites in vitro as well as in Alzheimer's disease brain. The enzyme is therefore regarded as an important candidate responsible for the progression of Alzheimer's disease. We and others have suggested that the enzyme has physiological roles in brain development and maturation. In this study, we investigated the exact distribution and developmental changes of the enzyme in cerebellum by immunohistochemistry and non-radioactive in situ hybridization. Immunohistochemistry demonstrated that Cdk5 was consistently expressed in the cerebellum at all developing stages. However, the subcellular localization of Cdk5 dramatically changed during maturation of the cerebellum. In the early neonatal stage, Cdk5 was strongly expressed in the cell bodies of neurones. With neuronal maturation Cdk5 immunoreactivity changed its subcellular localization from the cell body to axon. In terminally differentiated neurons, the immunoreactivity was only detected in the axon. These results suggest that subcellular localization of Cdk5 is strictly regulated and may play an important role in neuronal maturation.

Alzheimer Disease↗

PubMed2001

Subcellular redistribution of BAX during apoptosis induced by anticancer drugs.

BAX is the 192-amino acid, 21-kDa protein which is ubiquitously distributed in normal tissues and is regarded as a tumor suppressor sensitizing malignant cells to anticancer drugs. In spite of many studies, the molecular mechanism of BAX action is still obscure. In the present study subcellular BAX translocations in human colon adenocarcinoma COLO 205 cells exposed to various anticancer drugs [camptothecin (CPT), etoposide (ETO), staurosporine (STP), 2-chloro-2'-deoxyadenosine (2CdA) and nimesulide (NIM)] was examined. Cells were grown on coverslips under optimal conditions (10% FCS/DMEM) or were stimulated to apoptosis with the drugs examined. Laser scanning cytometry was applied for the quantitative analysis of BAX expression, and distribution in the cytoplasmic (BAX Cf) and nuclear (BAX Nf) area. BAX maximal pixel (BAX MP), the parameter corresponding to aggregation of BAX in the cell, was also measured. All examined drugs increased the number of cells with high BAX MP, reaching the peak at 60 min after drug administration. The most pronounced effect was in the case of 2CdA, CPT and STP. The increase in BAX MP was observed only when antibody recognizing the 43-61 amino acid sequence was used. When antibody binding the N-terminal epitope (11-30 amino acid sequence) was applied, the number of cells expressing high BAX MP significantly decreased. These results indicate that apoptotic stimuli delivered by anticancer drugs led to aggregation of BAX in cancer cells, which is dependent on BAX activation by its cleavage at the N-terminal epitope and exposure of the BH3 domain. It was shown that BAX Nf increased in cells treated with CPT, STP, ETO, 2CdA and NIM, whereas BAX Cf rose after STP and NIM. The increase in BAX Nf and, occurring in most treatments, the increase in the BAX Nf:Cf ratio indicates a BAX shift from the cytoplasm to the nucleus. Furthermore, staining with different antibodies showed that only the activated form of BAX was translocated to the nucleus. Immunoelectron microscopy revealed that CPT-induced apoptosis was associated with translocation of BAX from the cytosol to organellar membranes (mitochondrial, Golgi apparatus and endoplasmic reticulum) and via nuclear envelope pores to the nucleus, occurring within 60-180 min of cell exposure to the drug. The subcellular translocations of BAX preceded in time the appearance of morphological symptoms of apoptosis. In conclusion, (i) in spite of different molecular mechanisms of apoptosis induction by the anticancer drugs examined, BAX remains a common link in the chain of reactions leading to cell death, and (ii) BAX activation and subcellular translocations from the cytosol to organellar membranes and nucleus are key cellular responses to drugs bearing proapoptotic properties.

Adenocarcinoma↗

PubMed2005

Differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins S, M and E.

Post-translational modifications and correct subcellular localization of viral structural proteins are prerequisites for assembly and budding of enveloped viruses. Coronaviruses, like the severe acute respiratory syndrome-associated virus (SARS-CoV), bud from the endoplasmic reticulum-Golgi intermediate compartment. In this study, the subcellular distribution and maturation of SARS-CoV surface proteins S, M and E were analysed by using C-terminally tagged proteins. As early as 30 min post-entry into the endoplasmic reticulum, high-mannosylated S assembles into trimers prior to acquisition of complex N-glycans in the Golgi. Like S, M acquires high-mannose N-glycans that are subsequently modified into complex N-glycans in the Golgi. The N-glycosylation profile and the absence of O-glycosylation on M protein relate SARS-CoV to the previously described group 1 and 3 coronaviruses. Immunofluorescence analysis shows that S is detected in several compartments along the secretory pathway from the endoplasmic reticulum to the plasma membrane while M predominantly localizes in the Golgi, where it accumulates, and in trafficking vesicles. The E protein is not glycosylated. Pulse-chase labelling and confocal microscopy in the presence of protein translation inhibitor cycloheximide revealed that the E protein has a short half-life of 30 min. E protein is found in bright perinuclear patches colocalizing with endoplasmic reticulum markers. In conclusion, SARS-CoV surface proteins S, M and E show differential subcellular localizations when expressed alone suggesting that additional cellular or viral factors might be required for coordinated trafficking to the virus assembly site in the endoplasmic reticulum-Golgi intermediate compartment.

Animals↗

PubMed2002

Targeting tryptophan decarboxylase to selected subcellular compartments of tobacco plants affects enzyme stability and in vivo function and leads to a lesion-mimic phenotype.

Tryptophan decarboxylase (TDC) is a cytosolic enzyme that catalyzes an early step of the terpenoid indole alkaloid biosynthetic pathway by decarboxylation of L-tryptophan to produce the protoalkaloid tryptamine. In the present study, recombinant TDC was targeted to the chloroplast, cytosol, and endoplasmic reticulum (ER) of tobacco (Nicotiana tabacum) plants to evaluate the effects of subcellular compartmentation on the accumulation of functional enzyme and its corresponding enzymatic product. TDC accumulation and in vivo function was significantly affected by the subcellular localization. Immunoblot analysis demonstrated that chloroplast-targeted TDC had improved accumulation and/or stability when compared with the cytosolic enzyme. Because ER-targeted TDC was not detectable by immunoblot analysis and tryptamine levels found in transient expression studies and in transgenic plants were low, it was concluded that the recombinant TDC was most likely unstable if ER retained. Targeting TDC to the chloroplast stroma resulted in the highest accumulation level of tryptamine so far reported in the literature for studies on heterologous TDC expression in tobacco. However, plants accumulating high levels of functional TDC in the chloroplast developed a lesion-mimic phenotype that was probably triggered by the relatively high accumulation of tryptamine in this compartment. We demonstrate that subcellular targeting may provide a useful strategy for enhancing accumulation and/or stability of enzymes involved in secondary metabolism and to divert metabolic flux toward desired end products. However, metabolic engineering of plants is a very demanding task because unexpected, and possibly unwanted, effects may be observed on plant metabolism and/or phenotype.

Aromatic-L-Amino-Acid Decarboxylases↗

PubMed1980

Uptake and subcellular compartmentation of gibberellin a(1) applied to leaves of barley and cowpea.

The uptake and subcellular accumulation of gibberellin A(1) (GA(1)) by leaves and protoplasts of barley (cv. Numar) and cowpea (cv. Blackeye pea No. 5) were investigated.Uptake of GA(1) by cowpea leaves is optimal at pH 5.8 and occurs by a saturable, probably carrier-mediated process having a half-maximal velocity at 10 to 20 micromolar. Uptake by both barley and cowpea leaves is inhibited by low temperature (+4 C) and the metabolic inhibitors 2,4-dinitrophenol and azide and is stimulated by ATP. Mesophyll protoplasts isolated from leaves fed radioactive GA(1) retain 20 to 80% of the radioactivity incorporated by excised leaves.The subcellular localization of the [(3)H]GA was determined by lysing protoplasts and separating subcellular organelles by density gradient centrifugation. Less than 5% of the incorporated [(3)H]GA was found associated with chloroplasts, mitochondria, nuclei, or other organelles or membranes with densities in sucrose gradients greater than 1.15 grams per cubic centimeter. Fifty to 100% of the [(3)H]GA was found in vacuoles. Isolated vacuoles were judged to be free of contamination by cytoplasm using phosphoenolpyruvate carboxylase as a marker enzyme. Osmotic breakage of vacuoles or protoplasts released > 95% of the [(3)H]GA, suggesting that GA is associated with the vacuolar sap rather than with the tonoplast membrane.

Journal Article↗