Prediction of subcellular protein localization based on functional domain composition

Assigning subcellular localization (SL) to proteins is one of the major tasks of functional proteomics. Despite the impressive technical advances of the past decades, it is still time-consuming and laborious to experimentally determine SL on a high throughput scale. Thus, computational predictions are the preferred method for large-scale assignment of protein SL, and if appropriate, followed up by experimental studies. In this report, using a machine learning approach, the Nearest Neighbor Algorithm (NNA), we developed a prediction system for protein SL in which we incorporated a protein functional domain profile. The overall accuracy achieved by this system is 93.96%. Furthermore, comparisons with other methods have been conducted to demonstrate the validity and efficiency of our prediction system. We also provide an implementation of our Subcellular Location Prediction System (SLPS), which is available at http://pcal.biosino.org.     

Transmembrane domain dependent inhibitory function of FcγRIIB

FcγRIIB, the only inhibitory IgG Fc receptor, functions to suppress the hyper-activation of immune cells. Numerous studies have illustrated its inhibitory function through the ITIM motif in the cytoplasmic tail of FcγRIIB. However, later studies revealed that in addition to the ITIM, the transmembrane (TM) domain of FcγRIIB is also indispensable for its inhibitory function. Indeed, recent epidemiological studies revealed that a non-synonymous single nucleotide polymorphism (rs1050501) within the TM domain of FcγRIIB, responsible for the I232T substitution, is associated with the susceptibility to systemic lupus erythematosus (SLE). In this review, we will summarize these epidemiological and functional studies of FcγRIIB-I232T in the past few years, and will further discuss the mechanisms accounting for the functional loss of FcγRIIB-I232T. Our review will help the reader gain a deeper understanding of the importance of the TM domain in mediating the inhibitory function of FcγRIIB and may provide insights to a new therapeutic target for the associated diseases.     

Prediction of protein subcellular localization

Because the protein's function is usually related to its subcellular localization, the ability to predict subcellular localization directly from protein sequences will be useful for inferring protein functions. Recent years have seen a surging interest in the development of novel computational tools to predict subcellular localization. At present, these approaches, based on a wide range of algorithms, have achieved varying degrees of success for specific organisms and for certain localization categories. A number of authors have noticed that sequence similarity is useful in predicting subcellular localization. For example, Nair and Rost (Protein Sci 2002;11:2836-2847) have carried out extensive analysis of the relation between sequence similarity and identity in subcellular localization, and have found a close relationship between them above a certain similarity threshold. However, many existing benchmark data sets used for the prediction accuracy assessment contain highly homologous sequences-some data sets comprising sequences up to 80-90% sequence identity. Using these benchmark test data will surely lead to overestimation of the performance of the methods considered. Here, we develop an approach based on a two-level support vector machine (SVM) system: the first level comprises a number of SVM classifiers, each based on a specific type of feature vectors derived from sequences; the second level SVM classifier functions as the jury machine to generate the probability distribution of decisions for possible localizations. We compare our approach with a global sequence alignment approach and other existing approaches for two benchmark data sets-one comprising prokaryotic sequences and the other eukaryotic sequences. Furthermore, we carried out all-against-all sequence alignment for several data sets to investigate the relationship between sequence homology and subcellular localization. Our results, which are consistent with previous studies, indicate that the homology search approach performs well down to 30% sequence identity, although its performance deteriorates considerably for sequences sharing lower sequence identity. A data set of high homology levels will undoubtedly lead to biased assessment of the performances of the predictive approaches-especially those relying on homology search or sequence annotations. Our two-level classification system based on SVM does not rely on homology search; therefore, its performance remains relatively unaffected by sequence homology. When compared with other approaches, our approach performed significantly better. Furthermore, we also develop a practical hybrid method, which combines the two-level SVM classifier and the homology search method, as a general tool for the sequence annotation of subcellular localization.     

FAM92A1-289基因的真核表达载体构建和亚细胞定位

利用PCR方法扩增FAM92A1-289全长,经BamH I和Xho I酶切后连接入pEGFP-N1真核表达载体,构建pEGFP-N1-FAM92A1-289重组表达质粒,转染Hela细胞,利用荧光显微镜观察FAM92A1-289在细胞中的定位。经双酶切和核酸序列分析证实重组质粒包含有正确编码的FAM92A1-289读码框。荧光显微镜观察到空质粒pEGFP-N1转染后,整个细胞内弥散绿色荧光,而转染pEGFP-N1-FAM92A1-289重组载体后,可见绿色荧光分布于Hela细胞核中,显示FAM92A1-289定位于细胞核。成功构建人FAM92A1-289真核表达载体,FAM92A1-289定位于哺乳细胞的细胞核中。     

Expression of EGFP/SDCT1 fusion protein, subcellular localization signal analysis, tissue distribution and electrophysiological function study

A main pathway for energy ATP production inhuman body is by tricarboxylic acid cycle (Krebs cy-cle). Sodium-dependent dicarboxylate co-transporterprotein (SDCT, NaDC, NaC) is an organic aniontransporter protein family responsible for trans-mem- for 30 s, and extension 72℃ for 2 min; followed bybrane transport of Krebs cycle intermediate metabolite final extension 72℃ for 7 min. PCR products weresuch as succinate and citrate. They predominantly lo- …     

The glycine arginine‐rich domain of the RNA‐binding protein nucleolin regulates its subcellular localization

Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein‐protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid‐liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA‐RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.     

Phosphorylation of the N-terminal domain regulates subcellular localization and DNA binding properties of the peptidyl-prolyl cis/trans isomerase hPar14

Human parvulin 14 (hPar14) is a folding helper enzyme belonging to the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases). This enzyme is thought to play a role in cell-cycle and chromatin remodeling. Although hPar14 was nuclearly localized and bound to double-stranded DNA, the molecular basis of the subcellular localization and the functional regulation remained unknown.Here we show that subcellular localization and DNA-binding ability of hPar14 is regulated by posttranslational modification of its N-terminal domain. As proved by MALDI-TOF mass spectrometry and MS/MS fragmentation, hPar14 is phosphorylated at Ser19 in vitro and in vivo. In human HeLa cells the protein is most likely modified by casein kinase 2 (CK2). Phosphorylation of hPar14 is inhibited both in vitro and in vivo by 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), a specific inhibitor of CK2 activity. Mutation of Ser19 to Ala abolishes phosphorylation and alters the subcellular localization of hPar14 from predominantly nuclear to significantly cytoplasmic. Immunostaining shows that a Glu19 mutant of hPar14, which mimics the phosphorylated state of Ser19, is localized around the nuclear envelope, but does not penetrate into the nucleoplasm. In contrast to wild-type hPar14, the in vitro DNA-binding affinity of the Glu19 mutant is strongly reduced, suggesting that only the dephosphorylated protein is the active DNA-binding form of hPar14 in the nucleus.     

蛋白质亚细胞定位预测中的序列编码技术

蛋白质序列的编码是亚细胞定位预测问题中的关键技术之一。该文较为详细地介绍了目前已有的蛋白质序列编码算法;并指出了序列编码中存在的一些问题及可能的发展方向。     

小麦铝胁迫基因TaAIP的克隆表达分析及亚细胞定位

以小麦抗逆相关蛋白TaMAPK2作为诱饵,利用酵母双杂交系统进行筛库,获得互作蛋白TaAIP。氨基酸序列分析发现TaAIP具有wali保守区,并且与一些物种的铝诱导蛋白相似。实时荧光定量PCR分析显示,TaAIP基因受到铝、干旱以及高盐胁迫上调表达。半定量RT-PCR结果表明,TaAIP在小麦茎中表达,在根部、叶片以及花中没有表达。亚细胞定位实验发现,TaAIP定位在细胞膜上。这些结果为深入分析TaAIP的抗逆性作用机理打下基础。     

Nuclear localization of Chfr is crucial for its checkpoint function

Chfr, a checkpoint with FHA and RING finger domains, plays an important role in cell cycle progression and tumor suppression. Chfr possesses the E3 ubiquitin ligase activity and stimulates the formation of polyubiquitin chains by Ub-conjugating enzymes, and induces the proteasome-dependent degradation of a number of cellular proteins, including Plk1 and Aurora A. While Chfr is a nuclear protein that functions within the cell nucleus, how Chfr is localized in the nucleus has not been clearly demonstrated. Here, we show that nuclear localization of Chfr is mediated by nuclear localization signal (NLS) sequences. To reveal the signal sequences responsible for nuclear localization, a short lysine-rich stretch (KKK) at amino acid residues 257–259 was replaced with alanine, which completely abolished nuclear localization. Moreover, we show that nuclear localization of Chfr is essential for its checkpoint function but not for its stability. Thus, our results suggest that NLS-mediated nuclear localization of Chfr leads to its accumulation within the nucleus, which may be important in the regulation of Chfr activation and Chfr-mediated cellular processes, including cell cycle progression and tumor suppression.     

基于序列拓扑和二阶隐马尔可夫模型的跨膜蛋白亚细胞定位预测

现有蛋白质亚细胞定位方法针对水溶性蛋白质而设计,对跨膜蛋白并不适用。而专门的跨膜拓扑预测器,又不是为亚细胞定位而设计的。文章改进了跨膜拓扑预测器TMPHMMLoc的模型结构,设计了一个新的二阶隐马尔可夫模型;采用推广到二阶模型的Baum-Welch算法估计模型参数,并把将各个亚细胞位置建立的模型整合为一个预测器。数据集上测试结果表明,此方法性能显著优于针对可溶性蛋白设计的支持向量机方法和模糊k最邻近方法,也优于TMPHMMLoc中提出的隐马尔可夫模型方法,是一个有效的跨膜蛋白亚细胞定位预测方法。     

Light-dependent subcellular localization of nucleoside diphosphate kinase-1 in Neurospora crassa

Nucleoside diphosphate kinase (NDK) is a housekeeping enzyme localized in cellular organelles and distributed in various organs in prokaryotes and eukaryotes. In Neurospora crassa, NDK-1 is suggested to control catalases in response to heat, oxidative stress and light. In this study, we identified the presence of NDK-1 during most developmental stages in submerged mycelia, aerial hyphae, asexual conidia and perithecia, and the localization of it in soluble, mitochondrial, nuclear and membrane fractions in the mycelial cell. A light-dependent localization of NDK-1 was shown by Western blotting and immunohistochemical analysis using anti-NDK-1 antibody. In the mycelia, NDK-1 was compartmentalized on the plasma membrane in darkness, while it was relocated in the cytoplasm under light. These results suggest that NDK-1 protein was translocated from the plasma membrane to cytoplasm in response to light, and may interact with catalase.     

Subcellular localization of CIAPIN1.

Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) is a newly identified anti-apoptotic molecule. Our previous studies have demonstrated that CIAPIN1 is ubiquitously expressed in normal fetal and adult human tissues and confers multidrug resistance in gastric cancer cells, possibly by upregulating the expression of multidrug resistance gene 1 and multidrug resistance-related protein 1. However, fundamental biological functions of CIAPIN1 have not been elucidated. In this study, we first predicted the subcellular localization of CIAPIN1 with bioinformatic approaches and then characterized the intracellular localization of CIAPIN1 in both human and mouse cells by a combination of techniques including (a)immunohistochemistry and immunofluorescence, (b) His-tagged CIAPIN1 expression, and (c)subcellular fractionation and analysis of CIAPIN1 in the fractions by Western blotting. All methods produced consistent results; CIAPIN1 was localized in both the cytoplasm and the nucleus and was accumulated in the nucleolus. Bioinformatic prediction disclosed a putative nuclear localization signal and a putative nuclear export signal within both human and mouse CIAPIN1. These findings suggest that CIAPIN1 may undergo a cytoplasm-nucleus-nucleolus translocation.     

Predicting subcellular localization of proteins by hybridizing functional domain composition and pseudo-amino acid composition

Recent advances in large-scale genome sequencing have led to the rapid accumulation of amino acid sequences of proteins whose functions are unknown. Since the functions of these proteins are closely correlated with their subcellular localizations, many efforts have been made to develop a variety of methods for predicting protein subcellular location. In this study, based on the strategy by hybridizing the functional domain composition and the pseudo-amino acid composition (Cai and Chou [2003]: Biochem. Biophys. Res. Commun. 305:407-411), the Intimate Sorting Algorithm (ISort predictor) was developed for predicting the protein subcellular location. As a showcase, the same plant and non-plant protein datasets as investigated by the previous investigators were used for demonstration. The overall success rate by the jackknife test for the plant protein dataset was 85.4%, and that for the non-plant protein dataset 91.9%. These are so far the highest success rates achieved for the two datasets by following a rigorous cross validation test procedure, further confirming that such a hybrid approach may become a very useful high-throughput tool in the area of bioinformatics, proteomics, as well as molecular cell biology.     

Expression and subcellular localization of mechano-growth factor in osteoblasts under mechanical stretch

Mechano-growth factor (MGF) is a stretch sensitive factor in myocytes, and it might also be produced by other mechanocytes under mechanical stimulation. In this study, both the mRNA and protein expression of MGF were detected in stretched osteoblasts. Quantitative analysis showed that a cyclic stretching stimulation caused a quick and sharp increase of MGF mRNA and protein expression from a low basal level under no stretch; the mRNA and protein levels respectively peaked in 6 and 12 h to 5 and 5.2 fold over the basal level and returned to normal by 24 h. The subcellular distribution of MGF protein was revealed by immunofluorescence analysis to be restricted to the nucleus. We concluded that cyclic stretching stimulation could induce MGF expression in osteoblasts in a pulsing fashion; and the nuclear distribution of MGF suggested that MGF might act in mechanocytes as an autocrine growth factor.