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PubMed2006

Prediction of the subcellular location of apoptosis proteins.

Apoptosis proteins have a central role in the development and the homeostasis of an organism. These proteins are very important for understanding the mechanism of programmed cell death. The function of an apoptosis protein is closely related to its subcellular location. Based on the concept that the subcellular location of an apoptosis protein is mainly determined by its amino acid sequence, a new algorithm for prediction of the subcellular location of an apoptosis protein is proposed. By using of a distinctive set of information parameters derived from the primary sequence of 317 apoptosis proteins, the increment of diversity (ID), the sole prediction parameter, is calculated. The higher predictive success rates than the previous other algorithms is obtained by the jackknife tests using the expanded dataset. Our prediction results show that the local compositions of twin amino acids and hydropathy distribution are very useful to predict subcellular location of protein.

Algorithms↗

PubMed2006

Analysis of the influence of subcellular localization of the HIV Rev protein on Rev-dependent gene expression by multi-fluorescence live-cell imaging.

The human immunodeficiency virus Rev protein is a post-transcriptional activator of HIV gene expression. Rev is a nucleocytoplasmic shuttle protein that displays characteristic nuclear/nucleolar subcellular localization in various cell lines. Cytoplasmic localization of Rev occurs under various conditions disrupting Rev function. The goal of this study was to investigate the relationship between localization of Rev and its functional activity in living cells. A triple-fluorescent imaging assay, called AQ-FIND, was established for automatic quantitative evaluation of nucleocytoplasmic distribution of fluorescently tagged proteins. This assay was used to screen 500 rev genes generated by error-prone PCR for Rev mutants with different localization phenotypes. Activities of the Rev mutants were determined with a second quantitative, dual-fluorescent reporter assay. In HeLa cells, the majority of nuclear Rev mutants had activities similar to wild-type Rev. The activities of Rev mutants with abnormal cytoplasmic localization ranged from moderately impaired to nonfunctional. There was no linear correlation between subcellular distribution and levels of Rev activity. In astrocytes, nuclear Rev mutants showed similar impaired activities as the cytoplasmic wild-type Rev. Our data suggest that steady-state subcellular localization is not a primary regulator of Rev activity but may change as a secondary consequence of altered Rev function. The methodologies described here have potential for studying the significance of subcellular localization for functions of other regulatory factors.

Active Transport, Cell Nucleus↗

PubMed2001

Membrane-initiated Ca(2+) signals are reshaped during propagation to subcellular regions.

An important aspect of Ca(2+) signaling is the ability of cells to generate intracellular Ca(2+) waves. In this study we have analyzed the cellular and subcellular kinetics of Ca(2+) waves in a neuroendocrine transducer cell, the melanotrope of Xenopus laevis, using the ratiometric Ca(2+) probe indo-1 and video-rate UV confocal laser-scanning microscopy. The purpose of the present study was to investigate how local Ca(2+) changes contribute to a global Ca(2+) signal; subsequently we quantified how a Ca(2+) wave is kinetically reshaped as it is propagated through the cell. The combined kinetics of all subcellular Ca(2+) signals determined the shape of the total cellular Ca(2+) signal, but each subcellular contribution to the cellular signal was not constant in time. Near the plasma membrane, [Ca(2+)](i) increased and decreased rapidly, processes that can be described by a linear and exponential function, respectively. In more central parts of the cell slower kinetics were observed that were best described by a Hill equation. This reshaping of the Ca(2+) wave was modeled with an equation derived from a low-pass RC filter. We propose that the differences in spatial kinetics of the Ca(2+) signal serves as a mechanism by which the same cellular Ca(2+) signal carries different regulatory information to different subcellular regions of the cell, thus evoking differential cellular responses.

Animals↗

PubMed2001

Regulation of subcellular localization of alpha1-adrenoceptor subtypes.

Alpha1-adrenergic receptors (AR) are members of the superfamily of G protein-coupled receptors (GPCRs) which mediate the effects of the sympathetic nervous system. Alpha1-AR comprise a heterogeneous family of three distinct isoforms of alpha1A, alpha1B and alpha1D; however, very little is known about their difference in physiological role or regulation. We have recently observed a subtype-specific differences in subcellular localization of alpha1-ARs; thus, alpha1A-AR predominantly localize intracellularly, while alpha1B-AR on the cell surface. To examine the molecular mechanism for the subtype-specific differences in subcellular localization, we conducted a search for novel proteins that interact with the alpha1B-AR, specifically focusing on the carboxyl-terminal cytoplasmic domain. Using interaction cloning and biochemical techniques, we demonstrate that gC1q-R interacts with alpha1B-AR in vitro and in vivo through the specific site, and that in cells which co-express alpha1B-AR and gC1q-R, the subcellular localization of alpha1B-AR is markedly altered and its expression is down-regulated. These results suggest that gC1q-R plays a role in the regulation of the subcellular localization as well as the function of alpha1B-ARs.

Animals↗

PubMed2003

Subcellular localization of human glyceraldehyde-3-phosphate dehydrogenase is independent of its glycolytic function.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered a classical glycolytic protein involved exclusively in cytosolic energy production. However, recent evidence suggests that it is a multifunctional protein displaying diverse activities distinct from its conventional metabolic role. These new roles for GAPDH may be dependent on its subcellular localization, oligomeric state or on the proliferative state of the cell. GAPDH is encoded by a single gene without alternate splicing. The regulatory mechanisms are unknown through which an individual GAPDH molecule fulfills its non-glycolytic functions or is targeted to a specific intracellular localization. Accordingly, as a first step to elucidate these subcellular regulatory mechanisms, we examined the interrelationship between the intracellular expression of the GAPDH protein and its glycolytic function in normal human fetal and senior cells. GAPDH localization was determined by immunoblot analysis. Enzyme activity was quantitated by in vitro biochemical assay. We now report that the subcellular expression of GAPDH was independent of its classical glycolytic function. In particular, in both fetal and senior cells, considerable GADPH protein was present in intracellular domains characterized by significantly reduced catalysis. Gradient analysis indicated that this lower activity was not due to the dissociation of tetrameric GAPDH. These results suggest that human cells contain significant intracellular levels of enzymatically inactive GAPDH which is age-independent. The possibility is considered that the functional diversity of GAPDH may be mediated either by posttranslational alteration or by subcellular protein:protein and/or protein:nucleic acid interactions.

Cell Nucleus↗

PubMed2001

Subcellular localisation of cyclin B, Cdc2 and p21(WAF1/CIP1) in breast cancer. association with prognosis.

The heterodimeric cyclin B/Cdc2 protein kinase governs entry into mitosis, and can be negatively regulated through p53-mediated transcriptional induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). Ectopic expression of p21(WAF1/CIP1) in cultured cells has been shown previously to influence the subcellular distribution of the cyclin-dependent kinases (CDKs) including Cdc2. In this study, we have examined the subcellular localisation of Cdc2, cyclin B and p21(WAF1/CIP1) by immunohistochemistry in a well characterised series of primary breast cancers. Surprisingly, p21(WAF1/CIP1) was predominantly cytoplasmic in many of the tumours, where it was associated with high p53 levels; cytoplasmic p21(WAF1/CIP1) and high cyclin B levels were also significant predictors of poor prognosis. We conclude that breast tumorigenesis may be characterised by abnormalities in pathways determining not only levels of expression of key regulatory molecules, but also their subcellular localisation. Investigation of the subcellular distribution of cell cycle regulatory proteins, particularly p21(WAF1/CIP1), could provide valuable prognostic markers in breast cancer.

Adult↗

PubMed1985

Subcellular localization of renal kallikrein by ultrastructural immunocytochemistry.

The subcellular distribution of immunoreactive kallikrein was described in the rat nephron using ultrastructural immunocytochemistry. The renal tissue was fixed with a mixture of buffered picric acid-paraformaldehyde-glutaraldehyde and immunostained with the peroxidase-antiperoxidase method for the electron microscope with the following steps: antikallikrein antiserum, anti-IgG serum, peroxidase-antiperoxidase complex, 3-3' diaminobenzidine-H2O2, and post-staining with osmium tetroxide. Preabsorption of the primary antiserum with purified rat urinary kallikrein and substitution with normal serum were used as controls. As we have described previously, kallikrein was present exclusively in the connecting tubule cell of the distal nephron. Subcellularly, kallikrein was distributed in luminal membranes, basal membranes, rough endoplasmic reticulum, Golgi apparatus, and vesicles. The immunoreactive vesicles were present in the proximity of the Golgi apparatus and in the cytoplasm in the way between the Golgi and the luminal and basal plasma membranes. No immunostaining was observed in other subcellular components of the connecting tubule cell or in the other type of cell. With the description of kallikrein in subcellular organelles involved in the synthesis, processing, and transport of glycoproteins, we have advanced an hypothetical intracellular processing pathway for renal kallikrein.

Animals↗

PubMed2002

Differential subcellular localization of functionally divergent survivin splice variants.

Survivin is an inhibitor of apoptosis protein (IAP) that is markedly overexpressed in most cancers. We identified two novel functionally divergent splice variants, i.e. non-antiapoptotic survivin-2B and antiapoptotic survivin-deltaEx3. Because survivin-2B might be a naturally occurring antagonist of antiapoptotic survivin variants, we analyzed the subcellular distribution of these proteins. PSORT II analysis predicted a preferential cytoplasmic localization of survivin and survivin-2B, but a preferential nuclear localization of survivin-deltaEx3. GFP-tagged survivin variants confirmed the predicted subcellular localization and additionally revealed a cell cycle-dependent nuclear accumulation of survivin-deltaEx3. Moreover, a bipartite nuclear localization signal found exclusively in survivin-deltaEx3 may support cytoplasmic clearance of survivin-deltaEx3. In contrast to the known association between survivin and microtubules or centromeres during mitosis, no corresponding co-localization became evident for survivin-deltaEx3 or survivin-2B. In conclusion, our study provided data on a differential subcellular localization of functionally divergent survivin variants, suggesting that survivin isoforms may perform different functions in distinct subcellular compartments and distinct phases of the cell cycle.

Alternative Splicing↗

PubMed2000

Subcellular post-transcriptional targeting: delivery of an intracellular protein to the extracellular leaflet of the plasma membrane using a glycosyl-phosphatidylinositol (GPI) membrane anchor in neurons and polarised epithelial cells.

The effectiveness of viral vector-mediated gene transfer depends on the expression of therapeutic transgenes in the correct target cell types. So far, however, little attention has been given to targeted subcellular distribution of expressed transgenes. Targeting individual transgenes to particular subcellular compartments will provide various advantages in increasing the safety, efficacy, and specificity of viral vector-mediated gene delivery. Viruses normally hijack the cellular protein synthesis machinery for their own advantages. It is thus unknown whether cells infected with viral vectors will be able to target proteins to the correct subcellular organelles, or whether the subcellular targeting machinery would be selectively disrupted by viral infection. In this article we explored whether a herpes simplex virus type 1-derived vector could be used to deliver a transgene engineered to be targeted to the extracellular membrane of target cells. To do so we constructed a temperature-sensitive mutant HSV-1 vector, tsK-TT21 expressing a recombinant marker protein, tissue inhibitor of metalloproteinases (TIMP), linked to sequence encoding a signal for the addition of a glycosyl-phosphatidylinositol (GPI)-anchor within the endoplasmic reticulum. Our results demonstrate that HSV1-derived viral vectors can be used to target transgenes as GPI anchored proteins to the outside leaflet of plasma membranes, without disrupting the targeting machinery of host epithelial cells or neurons. This approach could then be used to target specific proteins to the cell membrane to modify cell-cell interactions, the function of specific plasma membrane proteins, or their interactions with other membrane proteins, and also to target a prodrug converting enzyme to the plasma membrane of target cells, therefore enhancing its cell killing effects.

Biotinylation↗

PubMed1967

Subcellular distribution of cytochrome c in rat liver. Methods for its extraction and purification.

1. A method for the extraction and purification of cytochrome c from rat liver is described. The method depends on multiple chromatography on Amberlite IRC-50 with elution with ammonium phosphate buffers of differing ionic composition and pH, interspersed with gel filtration with Sephadex G-25. Conditions leading to denaturation are avoided and the product is chromatographically pure. 2. The method may be used for the quantitative analysis of cytochrome c either in unfractionated liver or in subcellular fractions. 3. Two pools of cytochrome c were detected, one extractable at pH4.0 with distilled water and the other extracted from the residues of the first extraction with 0.15m-sodium chloride. 4. For subcellular distribution studies the liver was homogenized in 0.3m-sucrose and a nuclear fraction (washed thoroughly to remove trapped mitochondria), a mitochondrial fraction, a heavy microsomal fraction, a standard microsomal fraction and the cell sap were isolated. The mitochondrial fraction was subfractionated further by density-gradient centrifugation. Each fraction was analysed for protein, RNA, DNA, succinate-neotetrazolium oxidoreductase and glucose 6-phosphatase. 5. A total of 123mug. of cytochrome c was obtained/g. wet wt. of rat liver. 6. Values for the percentage subcellular distribution of cytochrome c are: nuclear fraction, 24.4; mitochondrial fraction, 57.2; heavy microsomal fraction, 5.2; standard microsomal fraction, 10.6; cell sap, 2.7. 7. Three out of the eight mitochondrial subfractions separated by gradient centrifugation contained 76% of the cytochrome c and 85% of the succinate-neotetrazolium oxidoreductase present in the mitochondrial fraction. 8. In unfractionated liver 94% of the cytochrome c was extracted at pH4.0 with water whereas in most of the subcellular fractions the corresponding value was approx. 75-80%.

Animals↗

PubMed1967

Assimilation of glucose carbon in subcellular rat brain particles in vivo and the problems of axoplasmic flow.

1. Rats were injected with [U-(14)C]glucose and the content of (14)C in proteins and lipids of the cerebral P(1) (;nuclear'), P(2) (;mitochondrial'), P(3) (;microsomal') and high-speed supernatant fractions was measured 7, 22 and 93hr. after injection of labelled glucose. 2. The crude brain mitochondrial fractions (P(2)) were subfractionated on continuous sucrose gradients (0.32-1.8m-sucrose) and the (14)C content of the proteins and lipids of about 20 subfractions was measured. 3. About 40-50% of the (14)C assimilated by brain proteins was found in the P(2) (;mitochondrial') fraction. About 68-70% of the (14)C assimilated by brain lipids was also recovered from the lipids of the P(2) fraction. 4. Between 22 and 93hr. after injection of [U-(14)C]glucose both the amount of (14)C in the protein of the P(2) (;mitochondrial') fraction and the specific activity of this protein increased. The specific activity of the protein of all other particulate fractions (P(1), P(2) and P(3)) and subfractions (obtained from sucrose-density-gradient subfractionation of fraction P(2)) when related to the specific activity of the high-speed supernatant protein also increased during 93hr. after injection of [U-(14)C]glucose. The amount of (14)C in the protein of the high-speed supernatant and the specific activity of this protein decreased during the same period. 5. The distribution of (14)C in the lipids of all subcellular particulate fractions remained unchanged during the period 22-93hr. after injection of [U-(14)C]glucose. 6. It was concluded that a diffusion occurs of some supernatant proteins into subcellular particulate matter of the cerebrum and no significant preference for any subcellular particulate matter was observed. The lipids occur in the cerebrum mainly in a non-diffusible state, which is consistent with the view that they form almost entirely a part of the structure of the cerebrum. 7. The data obtained do not lend further support to the concept of axoplasmic flow within the cerebrum or the concept of a one-directional flow of mitochondria or other subcellular particles within the cerebrum.

Journal Article↗

PubMedDate not supplied

Regional and subcellular distribution of soluble aminopeptidase in the human and the rat brain: a comparative study.

In an attempt to elucidate the cellular function of the soluble aminopeptidases, we have analysed their activity in several subcellular fractions (synaptosomal, mitochondrial, microsomal, nuclear and cytosolic fraction) and in different areas (amygdala, hypothalamus, hippocampus, striatum, frontal cortex, occipital cortex and parietal cortex) of the human and the rat brain. The enzymes assayed in this study were five cytosolic aminopeptidases identified inmammalian brain tissues: alanyl-aminopeptidase, arginyl-aminopeptidase, leucyl-aminopeptidase, pyroglutamyl-peptidase I and aspartyl-aminopeptidase. The regional comparative study revealed significantly higher activities of alanyl-aminopeptidase activity in the human brain, with arginyl-aminopeptidase activities being higher in the rat brain. In the subcellular study, while the alanyl- and arginyl-aminopeptidase activities were quite homogeneous in all the subcellular fractions, the leucyl-aminopeptidase, pyroglutamyl-peptidase I and aspartyl-aminopeptidase activities were significantly higher in the synaptosomal fraction. The differential distribution of these enzymes could suggest that these activities have different functions in the distinct subcellular structures of the human and the rat brain.

Aminopeptidases↗

PubMed2000

Identification of amino acid residues and protein kinases involved in the regulation of NFATc subcellular localization.

The subcellular localization of the transcription factor NFATc is tightly regulated by the calcium-regulated phosphatase calcineurin, which acts to directly dephosphorylate NFATc, causing its rapid translocation from the cytoplasm to the nucleus. The calcineurin-mediated nuclear localization of NFATc is opposed by poorly defined protein kinases that act either to directly antagonize nuclear import or, alternatively, to promote nuclear export. Here, we provide evidence that the cellular protein kinases JNK, ERK, p38, and CK2 (formerly casein kinase II) are involved in the regulation of NFATc subcellular localization. We show that JNK, ERK, and p38 physically associate with the NFATc N-terminal regulatory domain and can directly phosphorylate functionally important residues involved in regulating NFATc subcellular localization, namely Ser(172) and the conserved NFATc Ser-Pro repeats. Moreover, we found that overexpression of JNK, ERK, or p38 is able to block ionomycin-induced NFATc nuclear translocation, whereas treatment of cells with both PD98059 and SB202190, which inhibit MAPK/SAPK signaling pathways, is sufficient to trigger NFATc nuclear localization. Finally, we show that CK2 also binds the N terminus of NFATc and phosphorylates functionally important amino acid residues, including a conserved amino acid motif located downstream of each of the NFATc Ser-Pro repeats that appears to be important for regulating NFATc nuclear export. Collectively, these studies identify functionally important amino acid residues and protein kinases involved in the regulation of NFATc subcellular localization.

Amino Acid Sequence↗

PubMed2000

Characterization of the extra-large G protein alpha-subunit XLalphas. I. Tissue distribution and subcellular localization.

Our group previously described a new type of G protein, the 78-kDa XLalphas (extra large alphas) (Kehlenbach, R. H., Matthey, J., and Huttner, W. B. (1994) Nature 372, 804-809 and (1995) Nature 375, 253). Upon subcellular fractionation, XLalphas labeled by ADP-ribosylation with cholera toxin was previously mainly detected in the bottom fractions of a velocity sucrose gradient that contained trans-Golgi network and was differentially distributed to Galphas, which also peaked in the top fractions containing plasma membrane. Here, we investigate, using a new antibody specific for the XL domain, the tissue distribution and subcellular localization of XLalphas and novel splice variants referred to as XLN1. Upon immunoblotting and immunofluorescence analysis of various adult rat tissues, XLalphas and XLN1 were found to be enriched in neuroendocrine tissues, with a particularly high level of expression in the pituitary. By both immunofluorescence and immunogold electron microscopy, endogenous as well as transfected XLalphas and XLN1 were found to be predominantly associated with the plasma membrane, with only little immunoreactivity on internal, perinuclear membranes. Upon subcellular fractionation, immunoreactive XLalphas behaved similarly to Galphas but was differentially distributed to ADP-ribosylated XLalphas. Moreover, the bottom fractions of the velocity sucrose gradient were found to contain not only trans-Golgi network membranes but also certain subdomains of the plasma membrane, which reconciles the present with the previous observations. To further investigate the molecular basis of the association of XLalphas with the plasma membrane, chimeric proteins consisting of the XL domain or portions thereof fused to green fluorescent protein were analyzed by fluorescence and subcellular fractionation. In both neuroendocrine and non-neuroendocrine cells, a fusion protein containing the entire XL domain, in contrast to one containing only the proline-rich and cysteine-rich regions, was exclusively localized at the plasma membrane. We conclude that the physiological role of XLalphas is at the plasma membrane, where it presumably is involved in signal transduction processes characteristic of neuroendocrine cells.

Adenosine Diphosphate Ribose↗

PubMed2001

The N-methyl-D-aspartate receptor splice variant NR1-4 C-terminal domain. Deletion analysis and role in subcellular distribution.

The intracellular C-terminal domain of the N-methyl-d-aspartate receptor (NMDAR) subunits 1 (NR1) and 2 (NR2) are important, if not essential, to the process of NMDAR clustering and anchoring at the plasma membrane and the synapse. Eight NR1 splice variants exist, four of which arise from alternative splicing of the C-terminal exon cassettes. Alternative splice variants may display a differential ability to interact with synaptic anchoring proteins, and splicing of C-terminal exon cassettes may alter the mechanism(s) of subcellular localization and targeting. The NR1-4 isoform has a significantly different C-terminal composition than the prototypic NR1-1 isoform. Whereas the NR1-1 C terminus is composed of C0, C1, and C2 exon cassettes, the NR1-4 C terminus is composed of the C0 and C2' cassettes. In the present study, we address the importance of the NR1-4 C-terminal exon cassettes (C0C2') in subcellular localization in differentiated pheochromocytoma (PC12) cells, in organotypic cultures of dorsal root ganglia, and also in heterologous cells. NR1-4-green fluorescent protein chimeras were created with deletion of either C0, C2', or both cassettes to address their importance in subcellular distribution and cell surface expression of the NR1-4 subunit. These experiments demonstrate that the NR1-4 splice variant found predominantly in the spinal cord uses the C0 cassette, to a large degree, to organize the subcellular distribution of this receptor subunit. Although the role of the C2' subunit is less clear, it may be involved in subunit clustering. However, this clustering is not always as efficient as that attributed to C0 alone or to the natural combination of C0C2'. Finally, although an intact C-terminal domain is neither necessary for interaction with the NR2A subunit nor surface expression of the NR1-4 subunit, the C-terminal domain fragment alone blocks surface expression of native NR1-4, in a dominant negative fashion, when the two are coexpressed.

Alternative Splicing↗

PubMed2002

Expression analysis and subcellular distribution of the two G-protein regulators AGS3 and LGN indicate distinct functionality. Localization of LGN to the midbody during cytokinesis.

Activator of G-protein signaling 3 (AGS3) and LGN have a similar domain structure and contain four G-protein regulatory motifs that serve as anchors for the binding of the GDP-bound conformation of specific G-protein alpha subunits. As an initial approach to define further the different functional roles of AGS3 and LGN, we determined their expression profile and subcellular distribution. AGS3- and LGN-specific antisera indicated a widespread tissue distribution of LGN, whereas AGS3 is primarily enriched in brain. Brain punch biopsies of 13 discrete brain regions indicated that both AGS3 and LGN are expressed in all areas tested but are differentially regulated during development. LGN is expressed in neuronal, astroglial, and microglial cultures, whereas AGS3 expression is restricted to neurons. In primary neuronal cultures as well as in dividing cultures of PC12 cells, immunocytochemistry indicated distinct subcellular locations of AGS3 and LGN. The subcellular locations of the two proteins were differentially regulated by external stimuli and the cell cycle. In PC12 and COS7 cells, LGN moves from the nucleus to the midbody structure separating daughter cells during the later stages of mitosis, suggesting a role for G-proteins in cytokinesis. Thus, although AGS3 and LGN share a similar overall motif structure and both bind G-proteins, nature has endowed these proteins with different regulatory elements that allow functional diversity by virtue of tissue-specific expression and subcellular positioning.

Amino Acid Sequence↗

PubMed2005

Support vector machine-based method for subcellular localization of human proteins using amino acid compositions, their order, and similarity search.

Here we report a systematic approach for predicting subcellular localization (cytoplasm, mitochondrial, nuclear, and plasma membrane) of human proteins. First, support vector machine (SVM)-based modules for predicting subcellular localization using traditional amino acid and dipeptide (i + 1) composition achieved overall accuracy of 76.6 and 77.8%, respectively. PSI-BLAST, when carried out using a similarity-based search against a nonredundant data base of experimentally annotated proteins, yielded 73.3% accuracy. To gain further insight, a hybrid module (hybrid1) was developed based on amino acid composition, dipeptide composition, and similarity information and attained better accuracy of 84.9%. In addition, SVM modules based on a different higher order dipeptide i.e. i + 2, i + 3, and i + 4 were also constructed for the prediction of subcellular localization of human proteins, and overall accuracy of 79.7, 77.5, and 77.1% was accomplished, respectively. Furthermore, another SVM module hybrid2 was developed using traditional dipeptide (i + 1) and higher order dipeptide (i + 2, i + 3, and i + 4) compositions, which gave an overall accuracy of 81.3%. We also developed SVM module hybrid3 based on amino acid composition, traditional and higher order dipeptide compositions, and PSI-BLAST output and achieved an overall accuracy of 84.4%. A Web server HSLPred (www.imtech.res.in/raghava/hslpred/ or bioinformatics.uams.edu/raghava/hslpred/) has been designed to predict subcellular localization of human proteins using the above approaches.

Algorithms↗

PubMed2004

A comparative proteomic strategy for subcellular proteome research: ICAT approach coupled with bioinformatics prediction to ascertain rat liver mitochondrial proteins and indication of mitochondrial localization for catalase.

Subcellular proteomics, as an important step to functional proteomics, has been a focus in proteomic research. However, the co-purification of "contaminating" proteins has been the major problem in all the subcellular proteomic research including all kinds of mitochondrial proteome research. It is often difficult to conclude whether these "contaminants" represent true endogenous partners or artificial associations induced by cell disruption or incomplete purification. To solve such a problem, we applied a high-throughput comparative proteome experimental strategy, ICAT approach performed with two-dimensional LC-MS/MS analysis, coupled with combinational usage of different bioinformatics tools, to study the proteome of rat liver mitochondria prepared with traditional centrifugation (CM) or further purified with a Nycodenz gradient (PM). A total of 169 proteins were identified and quantified convincingly in the ICAT analysis, in which 90 proteins have an ICAT ratio of PM:CM>1.0, while another 79 proteins have an ICAT ratio of PM:CM<1.0. Almost all the proteins annotated as mitochondrial according to Swiss-Prot annotation, bioinformatics prediction, and literature reports have a ratio of PM:CM>1.0, while proteins annotated as extracellular or secreted, cytoplasmic, endoplasmic reticulum, ribosomal, and so on have a ratio of PM:CM<1.0. Catalase and AP endonuclease 1, which have been known as peroxisomal and nuclear, respectively, have shown a ratio of PM:CM>1.0, confirming the reports about their mitochondrial location. Moreover, the 125 proteins with subcellular location annotation have been used as a testing dataset to evaluate the efficiency for ascertaining mitochondrial proteins by ICAT analysis and the bioinformatics tools such as PSORT, TargetP, SubLoc, MitoProt, and Predotar. The results indicated that ICAT analysis coupled with combinational usage of different bioinformatics tools could effectively ascertain mitochondrial proteins and distinguish contaminant proteins and even multilocation proteins. Using such a strategy, many novel proteins, known proteins without subcellular location annotation, and even known proteins that have been annotated as other locations have been strongly indicated for their mitochondrial location.

Animals↗