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PubMed2002

Support vector machines for prediction of protein subcellular location by incorporating quasi-sequence-order effect.

Support Vector Machine (SVM), which is one class of learning machines, was applied to predict the subcellular location of proteins by incorporating the quasi-sequence-order effect (Chou [2000] Biochem. Biophys. Res. Commun. 278:477-483). In this study, the proteins are classified into the following 12 groups: (1) chloroplast, (2) cytoplasm, (3) cytoskeleton, (4) endoplasmic reticulum, (5) extracellular, (6) Golgi apparatus, (7) lysosome, (8) mitochondria, (9) nucleus, (10) peroxisome, (11) plasma membrane, and (12) vacuole, which account for most organelles and subcellular compartments in an animal or plant cell. Examinations for self-consistency and jackknife testing of the SVMs method were conducted for three sets consisting of 1,911, 2,044, and 2,191 proteins. The correct rates for self-consistency and the jackknife test values achieved with these protein sets were 94 and 83% for 1,911 proteins, 92 and 78% for 2,044 proteins, and 89 and 75% for 2,191 proteins, respectively. Furthermore, tests for correct prediction rates were undertaken with three independent testing datasets containing 2,148 proteins, 2,417 proteins, and 2,494 proteins producing values of 84, 77, and 74%, respectively.

Proteins↗

PubMed2003

Prediction and classification of protein subcellular location-sequence-order effect and pseudo amino acid composition.

Given a protein sequence, how to identify its subcellular location? With the rapid increase in newly found protein sequences entering into databanks, the problem has become more and more important because the function of a protein is closely correlated with its localization. To practically deal with the challenge, a dataset has been established that allows the identification performed among the following 14 subcellular locations: (1) cell wall, (2) centriole, (3) chloroplast, (4) cytoplasm, (5) cytoskeleton, (6) endoplasmic reticulum, (7) extracellular, (8) Golgi apparatus, (9) lysosome, (10) mitochondria, (11) nucleus, (12) peroxisome, (13) plasma membrane, and (14) vacuole. Compared with the datasets constructed by the previous investigators, the current one represents the largest in the scope of localizations covered, and hence many proteins which were totally out of picture in the previous treatments, can now be investigated. Meanwhile, to enhance the potential and flexibility in taking into account the sequence-order effect, the series-mode pseudo-amino-acid-composition has been introduced as a representation for a protein. High success rates are obtained by the re-substitution test, jackknife test, and independent dataset test, respectively. It is anticipated that the current automated method can be developed to a high throughput tool for practical usage in both basic research and pharmaceutical industry.

Algorithms↗

PubMed2006

Predicting protein subcellular location by fusing multiple classifiers.

One of the fundamental goals in cell biology and proteomics is to identify the functions of proteins in the context of compartments that organize them in the cellular environment. Knowledge of subcellular locations of proteins can provide key hints for revealing their functions and understanding how they interact with each other in cellular networking. Unfortunately, it is both time-consuming and expensive to determine the localization of an uncharacterized protein in a living cell purely based on experiments. With the avalanche of newly found protein sequences emerging in the post genomic era, we are facing a critical challenge, that is, how to develop an automated method to fast and reliably identify their subcellular locations so as to be able to timely use them for basic research and drug discovery. In view of this, an ensemble classifier was developed by the approach of fusing many basic individual classifiers through a voting system. Each of these basic classifiers was trained in a different dimension of the amphiphilic pseudo amino acid composition (Chou [2005] Bioinformatics 21: 10-19). As a demonstration, predictions were performed with the fusion classifier for proteins among the following 14 localizations: (1) cell wall, (2) centriole, (3) chloroplast, (4) cytoplasm, (5) cytoskeleton, (6) endoplasmic reticulum, (7) extracellular, (8) Golgi apparatus, (9) lysosome, (10) mitochondria, (11) nucleus, (12) peroxisome, (13) plasma membrane, and (14) vacuole. The overall success rates thus obtained via the resubstitution test, jackknife test, and independent dataset test were all significantly higher than those by the existing classifiers. It is anticipated that the novel ensemble classifier may also become a very useful vehicle in classifying other attributes of proteins according to their sequences, such as membrane protein type, enzyme family/sub-family, G-protein coupled receptor (GPCR) type, and structural class, among many others. The fusion ensemble classifier will be available at www.pami.sjtu.edu.cn/people/hbshen.

Amino Acids↗

PubMed1992

Subcellular localization of heparanase in human neutrophils.

The subcellular localization of a heparan sulfate degrading endoglycosidase, heparanase, was studied in human neutrophils. Unstimulated cells were disrupted by nitrogen cavitation and fractionated on a Percoll density gradient into three components, separating the plasma membranes, specific granules, and azurophilic granules. Heparanase activity was measured by gel filtration analysis of 35S-labeled degradation fragments released from subendothelial extracellular matrix (ECM) or produced during incubation with soluble, ECM-derived, heparan sulfate proteoglycans. Heparanase activity was found mainly in fractions containing the specific granules; this activity was inhibited by heparin. Freezing and thawing was not needed for recovery of the enzyme from the subcellular fraction, confirming previous data about its ready release. The mechanism of the ready release of heparanase from the specific granules requires further investigation.

Extracellular Matrix↗

PubMed1998

Subcellular localization of glycosphingolipids in human neutrophils.

The subcellular distribution of five major glycosphingolipids (GSLs) in human neutrophils was analyzed. The neutrophils were isolated from the blood of six donors and subdivided in three fractions containing the cell membranes, and the primary and the secondary granules, respectively. The separation was confirmed with antibodies detecting established subcellular fraction-specific molecules. The two main neutral GSLsGalbeta1-4Glcbeta1-1'Cer (lactosylceramide, LacCer) and nLc4Cer (paragloboside, PG) and the three gangliosides IV3NeuAcnLc4Cer (2-3SPG), IV6NeuAcnLc4Cer (2-6SPG), and VI3NeuAcnLc6Cer (2-3SnHC) were quantitated using the immunochemical digoxigenin (DIG) staining procedure. Secondary granules contained the highest amount of these GSLs. They are followed by the primary granules and the cell membranes. Based on this quantitation, we conclude that the majority of the GSLs of neutrophils occur intracellularly. These findings are in striking contrast to the general assumption of GSLs being mainly concentrated in the cell membrane.

Cell Membrane↗

PubMed2003

Subcellular alteration of glyceraldehyde-3-phosphate dehydrogenase in Alzheimer's disease fibroblasts.

The regulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been implicated both in age-related neurodegenerative disease and in apoptosis. Previous in vitro studies suggest an interaction between GAPDH and the beta-amyloid precursor protein (beta-APP), a protein directly involved in Alzheimer's disease (AD). New studies indicate that GAPDH is a multidimensional protein with diverse membrane, cytoplasmic, and nuclear functions; each is distinct from its role in glycolysis. The nuclear functions of GAPDH include a role in apoptosis that requires its translocation to the nucleus. Accordingly, beta-APP-GAPDH interactions, altering GAPDH structure in vivo, may affect energy generation, inducing hypometabolism, a characteristic AD phenotype. Because GAPDH is a multifunctional protein, pleiotropic effects may also occur in a variety of fundamental cellular pathways in AD cells. This may include unique GAPDH-RNA interactions. We report here the identification of a high-molecular-weight (HMW) GAPDH species present exclusively in the postnuclear fraction of AD cells. The latter is characterized by reduced GAPDH activity. The HMW GAPDH species was not detected in postnuclear age-matched control (AMC) fractions nor in AD whole-cell preparations. Each is characterized by normal GAPDH activity. By definition, the preparation of whole-cell extracts entails the destruction of subcellular structure. The latter findings indicate that the dissociation of the GAPDH protein from the HMW species restores its enzymatic activity. Thus, these results reveal a new, unique intracellular phenotype in AD cells. The functional consequences of subcellular alteration in GAPDH structure in AD cells are considered.

Age Factors↗

PubMed1982

Labeling of poly(A) associated RNA in synaptosomes and the other subcellular fractions of rat cerebral cortex in basal conditions and during training.

The labeling of total and poly(A) associated RNA from the subcellular fractions of cerebral cortex was studied after a 1-hour 3H-uridine pulse in control and trained rats. In the control animals, the fraction of newly synthesized brain cortex RNA which is found in synaptosomes is the minimal one. However, in synaptosomes the proportion of poly(A)+ RNA within newly synthesized RNA is maximal. Training of the rats does not influence total incorporation of label into RNA in the brain cortex and the distribution of newly synthesized RNA within the subcellular fractions. The proportions of poly(A)+ RNA within total labeled RNA in the various fractions are also unaffected.

Animals↗

PubMed1996

Expression and subcellular distribution of glutamate receptor subunits 2/3 in the developing cerebellar cortex.

The expression and subcellular location of glutamate receptor subunits 2&3 was investigated in the developing postnatal cerebellum. Immunoblotting revealed that glutamate receptor subunits 2/3 is expressed in an identical pattern of immunoreactive bands of approximately 108 kDa from postnatal day zero to adult animals. Light microscopy showed that within the cerebellar cortex, GluR 2/3 immunoreactivity was essentially confined to Purkinje neurons. Strong immunostaining could be observed at postnatal days 1-3 within Purkinje cell bodies and primary dendrites. With ongoing development, the cell body and an increasingly elaborate dendritic tree was outlined by immunoreaction product. In adult animals, staining of Purkinje cell dendrites was patchy, and staining intensity of the cell body, in particular, was greatly reduced. Ultrastructural analysis revealed that during early postnatal development, immunoreaction product was localized to the cell membrane, but was not confined to postsynaptic densities. From the second postnatal week, glutamate receptor subunits 2/3 immunoreactivity was largely restricted to postsynaptic densities. These observations reveal a developmentally regulated refinement of the subcellular distribution of defining subunits of the AMPA-type glutamate receptor. The presence of membrane bond receptors prior to the formation of synapses also provides a rationale for the known transmitter-mediated modulation of Purkinje cell dendritogenesis.

Age Factors↗

PubMed2004

Is the light chain subcellular localization an important factor in botulinum toxin duration of action?

Botulinum neurotoxins (BoNT) are therapeutic proteins that are specific, potent, and effective. They are highly specific in binding to motor neurons but do not bind to other non-neuronal cells. These proteins are zinc-dependent endopeptidases that inhibit exocytosis by specific cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein-receptor) proteins involved in vesicle docking and fusion. The therapeutic effect of BoNT/A in humans lasts from 3 to 12 months, depending upon the condition treated. Data from animal and cell culture models suggests that the long-lasting duration of inhibition of neurotransmitter release induced by BoNT/A maybe due to the persistence of the endopeptidase activity of the light chain (LC/A) in cells, interactions of the cleaved substrates, and/or the response of the nerve to the temporary disruption of communication with its target tissue. We have analyzed the subcellular localization of the light chains from serotypes A, B, and E and have demonstrated that each light chain displays a distinct distribution within cells. LC/A localizes at the plasma membrane, LC/B is dispersed throughout the cell including the nucleus, and LC/E is mainly cytosolic. Localization is similar in non-neuronal cell lines, suggesting that the signals involved in proper subcellular localization are within the LC sequences and the moiety the light chain interacts with is present in both neuronal and non-neuronal cells.

Amino Acid Sequence↗

PubMed2005

Distribution of conjugated linoleic acid in total and subcellular fractions from normal and cancerous parts of human testes.

The objective of the present study was to examine differences in the fatty acid composition of subcellular fractions from normal and cancerous parts of human testes. The conjugated linoleic acid (CLA) content was significantly higher in total testicular carcinoma (TC), but significantly lower in the mitochondrial fraction of TC in comparison to normal testicular tissue. The subcellular distribution pattern of CLA was similar to that of monounsaturated fatty acids, but different to that of 18:2n-6 (linoleic acid), underlining the different physiological properties of CLA and 18 : 2n-6. Because polyunsaturated fatty acids (PUFAs) have been suggested to have an effect on cancer risk and previous research has found that CLA inhibits the metabolism of 18 : 2n-6 into 20 : 4n-6, the contents of n-6 and n-3 PUFAs were determined. Significant differences were observed for 18 : 2n-6, 18 : 3n-3, 20 : 5n-3, and 22 : 6n-3, with 18 : 2n-6, 18 : 3n-3, and 20 : 5n-3 contents being higher and 22 : 6n-3 content being lower in TC than in normal testicular tissue. These results indicate a changed availability of substrates for the cyclooxygenase (COX) or lipooxygenase (LOX) pathways generating eicosanoids. Although not statistically significant, the reduced content of 20 : 4n-6 shown in this study might be due to an increased metabolism of this fatty acid into eicosanoids.

Cell Fractionation↗

PubMed1987

Nitroxides as potential contrast enhancing agents for MRI application: influence of structure on the rate of reduction by rat hepatocytes, whole liver homogenate, subcellular fractions, and ascorbate.

With an eye toward the development of nitroxides as potential contrast enhancing agents for MRI applications, we have compared the rates of reduction of 24 nitroxides of diverse structures by rat whole liver homogenate, hepatocytes, subcellular fractions, and ascorbate (10 eq excess). Our results indicate that five-membered ring nitroxides and alpha-carboxy alpha-aryl tert-butyl nitroxides are significantly more resistant toward bioreduction than six-membered ring and heterocyclic-substituted nitroxides in all systems. In the case of six-membered ring nitroxides the presence of either negatively charged or positively charged groups increases the susceptibility toward ascorbate reduction. The presence of carboxylate groups tends to enhance the resistance of all the nitroxides toward reduction by liver homogenate, hepatocytes, and subcellular fractions. trans-Azethoxyl nitroxides exhibited the best overall resistance toward bioreduction. The T1 relaxants of selected nitroxides were measured and found to be similar.

Animals↗

PubMed1989

Proteins of muscle subcellular fractions in Duchenne progressive muscular dystrophy stained with "stains-all" cationic carbocyanine dye and with Coomassie Blue.

The protein compositions of subcellular fractions of muscle obtained from 17 Duchenne dystrophy patients, 15 disease controls (10 different primary myopathies, 5 spinal muscular atrophy patients), and 10 normals were examined by polyacrylamide gel electrophoresis. The gels were stained with Coomassie Brilliant Blue and with "Stains-all," which stains calcium-binding proteins, sialic acid-rich glycoproteins, and phosphoproteins. In muscle membrane fractions of Duchenne dystrophy patients there was a marked reduction in the concentrations of calsequestrin and a 39 kDa protein that stained blue with "Stains-all." There were changes in the proteins of all subcellular fractions of Duchenne's patients; some of these changes appear to be specific for Duchenne dystrophy (DD). There was no apparent correlation between the protein changes observed on acrylamide gels and the age of the patients, the duration of the disease, the degree of disability, or activity of creatine kinase. A decreased level of calsequestrin in DD sarcoplasmic reticulum may contribute to an increased level of free calcium seen in muscle from these patients.

Calcium-Binding Proteins↗

PubMed2004

Convenient and versatile subcellular extraction procedure, that facilitates classical protein expression profiling and functional protein analysis.

Standardized sample preparation to reduce proteome complexity facilitates subsequent proteome analysis. Here we describe a robust sequential extraction method that enables simple fractionation of proteins in their native state according to their subcellular localization, yielding four subproteomes enriched in (a) cytosolic; (b) membrane and membrane organelle-localized; (c) soluble and DNA-associated nuclear and (d) cytoskeletal proteins. Efficiency and selectivity is demonstrated by morphological-, two-dimensional electrophoresis image-, immunological- as well as enzymatic-analysis. In pilot studies, subcellular redistribution of regulatory proteins was successfully measured.

Blotting, Western↗

PubMed2006

GNBSL: a new integrative system to predict the subcellular location for Gram-negative bacteria proteins.

This paper proposes a new integrative system (GNBSL--Gram-negative bacteria subcellular localization) for subcellular localization specifized on the Gram-negative bacteria proteins. First, the system generates a position-specific frequency matrix (PSFM) and a position-specific scoring matrix (PSSM) for each protein sequence by searching the Swiss-Prot database. Then different features are extracted by four modules from the PSFM and the PSSM. The features include whole-sequence amino acid composition, N- and C-terminus amino acid composition, dipeptide composition, and segment composition. Four probabilistic neural network (PNN) classifiers are used to classify these modules. To further improve the performance, two modules trained by support vector machine (SVM) are added in this system. One module extracts the residue-couple distribution from the amino acid sequence and the other module applies a pairwise profile alignment kernel to measure the local similarity between every two sequences. Finally, an additional SVM is used to fuse the outputs from the six modules. Test on a benchmark dataset shows that the overall success rate of GNBSL is higher than those of PSORT-B, CELLO, and PSLpred. A web server GNBSL can be visited from http://166.111.24.5/webtools/GNBSL/index.htm.

Amino Acid Sequence↗

PubMed1983

Effects of androgens on the ultrastructure and subcellular zinc distribution in the prostatic epithelium of castrated rats.

Sprague Dawley rats were maintained on testosterone propionate or 5 alpha-dihydrotestosterone for 3 days following bilateral orchidectomy for a 7-day period. Ultrastructural examination showed only partial recovery of the prostatic epithelium with testosterone propionate while 5 alpha-dihydrotestosterone caused the lateral and ventral lobes to revert to the appearance of control tissues. The latter metabolite induced greater stimulation of the prostate evidenced by increased mitotic division of the epithelial cells and an increased number of basal cells exhibiting ciliary formation was observed. Zinc concentrations in subcellular regions of both lateral and ventral prostate lobes were affected by the two androgens. Testosterone propionate was most effective in elevating zinc in the lateral lobe, particularly within the secretory components. In the ventral lobe both androgens caused an increase in subcellular zinc concentrations above control levels. The increase of nuclear and nucleolar zinc was related to the increase in nuclear activity and cellular response to the androgen administration.

Androgens↗

PubMed1987

Subcellular compartmentalization of calcium-dependent and calcium-independent neutral proteases in brain.

In the present experiments, we studied the subcellular distribution of three types of extralysosomal, neutral proteolytic activities in rat telencephalon: (1) nonthiol proteases (NTP), (2) thiol proteases (TP), and (3) calcium-activated thiol proteases (calpains I and II). Subcellular fractionation was performed by using conventional differential and sucrose-gradient centrifugation techniques. The only significant proteolytic activity detected in crude homogenates could be assigned to calpain II, the high-threshold calcium-activated protease. Within the primary fractions prepared from the homogenates, the highest levels of calpain II were found in S3, or the soluble cytoplasmic fraction. Significant activity of the enzyme was also present in P2, the crude mitochondrial/synaptosomal fraction. In contrast, the specific activity of calpain I was greatest in P2 with somewhat lesser enzymatic activity in P1 and S3. Most of the calpain I in P2 was recovered after differential centrifugation through sucrose gradients and lysis of the resultant subfractions. In marked contrast, only a small percentage of the calpain II activity was recovered in the gradient bands. In all, calpain II appears to be predominantly localized in the soluble cytoplasmic compartment while the greatest concentrations of calpain I are found in the soluble components of small glial and neuronal processes (pinched off during homogenization) that constitute the P2 fraction. The highest specific activity of the calcium-independent proteases was obtained in P3, a fraction essentially devoid of calpain, with a secondary peak in P2. Subfractionation of P2 revealed that calcium-independent TP in P2 was associated with mitochondria while the calcium-independent NTP was more uniformly distributed across myelin, synaptosomes, and mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Sigma-1 and sigma-2 sites in rat brain: comparison of regional, ontogenetic, and subcellular patterns.

Radioligand binding assay conditions were established for the selective labeling of sigma-1 and sigma-2 sites in membrane homogenates of rat brain. Selective sigma-1 assays were conducted using 5 nM(+)[3H]SKF-10,047 in the presence of 300 nM dizocilpine (MK-801). Selective sigma-2 assays were conducted using 5 nM [3H]DTG in the presence of 1 microM (+)SKF-10,047. Distributions of sigma-1 and sigma-2 binding among brain regions were found to differ. While the brain stem yields the highest level of sigma-1 binding, it yields among the lowest levels of sigma-2 binding. The reverse is true in hippocampal membranes. Different ontogenetic patterns were also observed. Sigma-2 binding decreases substantially during brain development, whereas sigma-1 binding does not vary significantly. Patterns of distribution among subcellular fractions of rat brain homogenates were found to be similar. Both sigma-1 and sigma-2 sites are most enriched in microsomal fractions, and neither is enriched in synaptosomal or mitochondrial fractions. The present results suggest that sigma-1 and sigma-2 sites are distinct entities; they do not appear to be located on a common macromolecule, and they do not represent two different affinity states of a single type of binding site. While the precise subcellular locations of sigma-1 and sigma-2 sites remain to be determined, we conclude that localization of either type of binding site to synaptic regions of plasma membrane or to mitochondria is highly unlikely.

Animals↗

PubMed1995

Increase in synaptic vesicle proteins in synapsin-transfected NG108-15 cells: a subcellular fractionation study.

We have examined the subcellular distribution of synapsins and synaptophysin in density gradients from synapsin- and vector-transfected NG108-15 cells, since we recently found that transfection of synapsin IIb cDNA into neuroblastoma x glioma hybrid cells (NG108-15) resulted in cell lines that had a more neuronal phenotype than controls. The increase in synapsins and synaptophysin in the transfected cells was maximal in the region of the gradient containing small synaptic vesicles. The transferrin receptor, a marker for early endosomes, did not increase in the synapsin-containing fractions in the transfected cells. Secretogranin I, a soluble protein stored in and secreted from large dense cored vesicles, showed a very pronounced increase in the dense regions of gradients from transfected cells. These subcellular fractionation data suggest a possible role for the synapsins in the regulation of synaptic vesicle function.

Animals↗