Essential proteins identification method based on four-order distances and subcellular localization information

Essential proteins are inseparable in cell growth and survival. The study of essential proteins is important for understanding cellular functions and biological mechanisms. Therefore, various computable methods have been proposed to identify essential proteins. Unfortunately, most methods based on network topology only consider the interactions between a protein and its neighboring proteins, and not the interactions with its higher-order distance proteins. In this paper, we propose the DSEP algorithm in which we integrated network topology properties and subcellular localization information in protein–protein interaction(PPI) networks based on four-order distances, and then used random walks to identify the essential proteins. We also propose a method to calculate the finite-order distance of the network, which can greatly reduce the time complexity of our algorithm. We conducted a comprehensive comparison of the DSEP algorithm with 11 existing classical algorithms to identify essential proteins with multiple evaluation methods. The results show that DSEP is superior to these 11 methods.

Prediction of Subcellular Localization of Eukaryotic Proteins Using Position-Specific Profiles and Neural Network with Weighted Inputs

Subcellular location is one of the key biological characteristics of proteins. Position-specific profiles （PSP） have been introduced as important characteristics of proteins in this article. In this study, to obtain position-specific profiles, the Position Specific lterative-Basic Local Alignment Search Tool （PSI-BLAST） has been used to search for protein sequences in a database. Position-specific scoring matrices are extracted from the profiles as one class of characteristics. Four-part amino acid compositions and lst-7th order dipeptide compositions have also been calculated as the other two classes of characteristics. Therefore, twelve characteristic vectors are extracted from each of the protein sequences. Next, the characteristic vectors are weighed by a simple weighing function and inputted into a BP neural network predictor named PSP-Weighted Neural Network （PSP-WNN）. The Levenberg-Marquardt algorithm is employed to adjust the weight matrices and thresholds during the network training instead of the error back propagation algorithm. With a jackknife test on the RH2427 dataset, PSP-WNN has achieved a higher overall prediction accuracy of 88.4% rather than the prediction results by the general BP neural network, Markov model, and fuzzy k-nearest neighbors algorithm on this dataset. In addition, the prediction performance of PSP-WNN has been evaluated with a five-fold cross validation test on the PK7579 dataset and the prediction results have been consistently better than those of the previous method on the basis of several support vector machines, using compositions of both amino acids and amino acid pairs. These results indicate that PSP-WNN is a powerful tool for subcellular localization prediction. At the end of the article, influences on prediction accuracy using different weighting proportions among three characteristic vector categories have been discussed. An appropriate proportion is considered by increasing the prediction accuracy.

Activities, Quantitative Changes and Subcellular Localization of α-Amylase During Development of Apple Fruit

Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuberous roots, and fleshy fruit development. α_Amylase is considered as one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was often shown extrachloroplastic in living cells. The present experiment showed that α_amylase activity was progressively increasing concomitantly with the decreasing starch concentrations during the development of apple ( Malus domestica Borkh cv. Starkrimson) fruit. The apparent amount of α_amylase assessed by Western blotting also increased during the fruit development, which is consistent with the seasonal changes in the enzyme activity. The enzyme subcellular_localization studies via immunogold electron_ microscopy technique showed that α_amylase visualized by gold particles was predominantly located in plastids, but the gold particles were scarcely found in other subcellular compartments. A high density of the enzyme was observed at the periphery of starch granules during the middle and late developmental stages. These data proved that the enzyme is compartmented in its functional sites in the living cells of the fruit. The predominantly plastid_distributed pattern of α_amylase in cells was shown unchanged throughout the fruit development. The density of gold particles (α_amylase) in plastids was increasing during the fruit development, which is consistent with the results of Western blotting. So it is considered that α_amylase is involved in starch hydrolysis in plastids of the fruit cells.

Subcellular Localization of Aquaporins OsPIP2-6 in Rice

[Objective] This study was to explore the subcellular localization of aquaporins OsPIP2-6 in rice. [Method] A key rice aquaporins gene OsPIP2-6 was cloned and used for construction of a transient expression vector,which was then transformed into onion epidermis via particle bombardment for confocal microscopy analysis using YFP gene as a reporter gene. [Result] The results showed that rice aquaporins OsPIP2-6 was mainly located in the plasma membrane. [Conclusion] Our results provided theoretical basis for further understanding plant aquaporins.

Construction of a fusion protein expression vector MK-EGFP and its subcellular localization in different carcinoma cell lines

AIM： To construct an expression plasmid encoding human wild-type midkine （MK） and enhanced green fluorescence protein （EGFP） fusion protein （MK-EGFP）, and to analyze the subcellular localization of MK in different cardnoma cell lines. METHODS： Two kinds of MK coding sequences with or without signal peptide were cloned into plasmid pEGFP-N2, and the recombinant plasmids constructed were introduced into HepG2, MCF7 and DU145 cells, respectively, by transfection. With the help of laser scanning confocal microscopy, the expression and subcellular localization of MK-GFP fusion protein could be detected. RESULTS： Compared with the GFP control, in which fluorescence was detected diffusely over the entire cell body except in the nucleolus, both kinds of fusion protein MK-GFP were localized exclusively to the nucleus and accumulated in the nucleolus in the three kinds of cancer cell lines. CONCLUSION： This study reveals the specific nucleolar translocation independent of signal peptide, which may be involved in the mechanism that MK works. It provides valuable evidence for further study on the functions of MK in nucleus and its possible mechanisms, in which ribosomal RNA transcription and ribosome assembly are involved.

Analysis of Specific Binding and Subcellular Localization of Wheat ERF Transcription Factor W17

The study aims to detect the subcellular localization of ERF （ethylene-responsive element binding factor） transcription factor W17 protein, the interaction between W 17 and cis-acting regulatory elements GCC-box and DRE in vitro, the binding and transactivating ability in vivo, and the role of W17 in higher plant stress-signal pathway. Recombinant plasmid W17/163hGFP was introduced into onion epidermal cells by the particle bombardment method with a PDS 1000/He. Transformed cells were incubated for 24 h at 22℃ in the dark and green fluorescence was monitored under a confocal microscope. The gene W17 was fused N-terminus of GST （glutathione-S-transferase） in prokaryotic expression vector pGEX-4T-1 and then transformed into E. coli strain BL21 （DE3）. IPTG （0.5 mmol L-1） was added to induce the expression of recombinant GST/W17 for 3 h. The fused proteins were purified by GST purification columns, and then subjected to gel retardation assay with a 32p-labeled GCC or DRE sequence. The different reporter and effector plasmids were introduced into tobacco leaves through agroinfiltration, then transformed leaves stained by X-Gluc, faded with 75% alcohol and monitored under a Stereozooming microscope. The GFP fused with W17 protein was localized in the nuclei; SDS-PAGE assay demonstrated that the fused protein GST/W17 could be induced and purified with molecular weight at around 42.2 kD under the induction of IPTG. Purified fused protein was able to specifically bind to both the wild-type GCC-box and DRE element, but had no interaction with either the mutant DRE or GCC-box; W17 protein can bind to GCC-box and transactive downstream GUS gene in vivo. W17 can localize into the nuclei, and it may be involved not only in biotic stresses controlled by GCC-box, but also in abiotic stresses （e.g., salt-） induced signaling pathway.

Subcellular localization of alpha-synuclein aggregates and their interaction with membranes

For more than a decade numerous evidence has been reported on the mechanisms of toxicity of α-synuclein（αS） oligomers and aggregates in α-synucleinopathies.These species were thought to form freely in the cytoplasm but recent reports of αS multimer conformations when bound to synaptic vesicles in physiological conditions,have raised the question about where αS aggregation initiates.In this review we focus on recent literature regarding the impact on membrane binding and subcellular localization of αS toxic species to understand how regular cellular function of αS contributes to pathology.Notably αS has been reported to mainly associate with specific membranes in neurons such as those of synaptic vesicles,ER/Golgi and the mitochondria,while toxic species of αS have been shown to inhibit,among others,neurotransmission,protein trafficking and mitochondrial function.Strategies interfering with αS membrane binding have shown to improve αS-driven toxicity in worms and in mice.Thus,a selective membrane binding that would result in a specific subcellular localization could be the key to understand how aggregation and pathology evolves,pointing out to αS functions that are primarily affected before onset of irreversible damage.

Influence of RNA interference on the mitochondrial subcellular localization of alpha-synuclein and on the formation of Lewy body-like inclusions in the cytoplasm of human embryonic kidney 293 cells induced by the overexpression of alphasynuclein

The specific and effective a-synuclein RNA interference （RNAi） plasmids, and the a-synuclein-pEGFP recombinant plasmids were co-transfected into human embryonic kidney 293 （HEK293） cells using the lipofectamine method. Using an inverted fluorescence microscope, a-synuclein proteins were observed to aggregate in the cytoplasm and nucleus. Wild-type a-synuclein proteins co-localized with mitochondria. Hematoxylin-eosin staining revealed round eosinophilic bodies （Lewy body-like inclusions） in the cytoplasm of some cells transfected with a-synuclein-pEGFP plasmid. However, the formation of Lewy body-like inclusions was not observed following transfection with the RNAi pSYN-1 plasmid. RNAi blocked Lewy body-like inclusions in the cytoplasm of HEK293 cells induced by wild-type a-synuclein overexpression, but RNAi did not affect the subcellular localization of wild-type a-synuclein in mitochondria.

An efficient transient gene expression system for protein subcellular localization assay and genome editing in citrus protoplasts

Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.

MORN motifs in plant PIPKs are involved in the regulation of subcellular localization and phospholipid binding

Multiple repeats of membrane occupation and recognition nexus (MORN) motifs were detected in plant phosphatidylinositl monophosphate kinase (PIPK), a key enzyme in PI-signaling pathway. Structural analysis indicates that all the MORN motifs (with varied numbers at ranges of 7-9), which shared high homologies to those of animal ones, were located at N-terminus and sequentially arranged, except those of OsPIPK1 and AtPIPK7, in which the last MORN motif was separated others by an -100 amino-acid "island" region, revealing the presence of two kinds of MORN arrangements in plant PIPKs. Through employing a yeast-based SMET (sequence of membrane-targeting) system, the MORN motifs were shown being able to target the fusion proteins to cell plasma membrane, which were further confirmed by expression of fused MORN-GFP proteins. Further detailed analysis via deletion studies indicated the MORN motifs in OsPIPK 1, together with the 104 amino-acid "island" region are involved in the regulation of differential subcellular localization, i.e. plasma membrane or nucleus, of the fused proteins. Fat Western blot analysis of the recombinant MORN polypeptide, expressed in Escherichia coli, showed that MORN motifs could strongly bind to PA and relatively slightly to PI4P and PI(4,5)P2. These results provide informative hints on mechanisms of subcellular localization, as well as regulation of substrate binding, of plant PIPKs.

Effect of Subcellular Localization of P21 on Proliferation and Apoptosis of HepG2 Cells

This study examined the effect of subcellular localization of P21 on the proliferation and apoptosis of HepG2 cells. The coding genes of the wild and the mutant P21 were amplified by mega primer PCR from the plasmid pCEP-WAF1 which contains human P21 cDNA in the nuclear localizational signal （NLS） sequence, and then inserted into the eukaryotic expression vector pDsRedl-C1. The recombinants were transfected into HepG2 cells. The transcription and expression of P21 were determined by RT-PCR and fluorescence microscopy. The cell proliferation was measured by MTT, and the cell cycle and apoptosis of HepG2 cells by flow cytometry. The results of restriction analysis, DNA sequencing and fluorescence microscopy confirmed the construction of the wild and the mutant P21 in the eukaryotic expression plasmid. The plasmid containing the mutant P21 was found to accelerate cell proliferation and the wild P21 plasmid to inhibit cell proliferation. Cell cycle analysis showed that the cell ratio of GeYG1 in the wild type group was significantly increased as compared with that in the mutant type group, and cell apoptosis analysis revealed that the apoptosis rate in the wild type group was much higher than that in the mutant type group. It was concluded that the subcellular localization of P21 may contribute to the develonment of heoatic cancer.

Subcellular Localization of APX1 Protein in Arabidopsis thaliana

To predict the subcellular location of ascorbate peroxidase 1 （ APX1 ） protein in Arabidopsis thaliana, a plasmid expressing APXl-enbance green fluores- cent protein （EGFP） fusion protein was constructed, transformed into Agrobacterium tumefaciens GV3101, and transiently expressed in tobacco leaves. The localization of APX1 protein in the cells was determined by detecting the distribution of the fusion protein in tobacco epidermal cells. The results showed APX1 protein was mainly distributed in the cytoplasm of the plant.

Glucose regulates LXRα subcellular localization and function in rat pancreatic β-cells

Liver X receptors(LXRs)are members of the nuclear receptor superfamily,which have been implicated in lipid ho-meostasis and more recently in glucose metabolism.Here,we show that glucose does not change LXRα protein level,but affects its localization in pancreatic β-cells.LXRα is found in the nucleus at 8 mM glucose and in the cytoplasm at4.2 mM.Addition of glucose translocates LXRα from the cytoplasm into the nucleus.Moreover,after the activation ofLXR by its synthetic non-steroidal agonist(T0901317),insulin secretion and glucose uptake are increased at 8 mM anddecreased at 4.2 mM glucose in a dose-dependent manner.Furthermore,at low glucose condition,okadaic acid reversedLXRα effect on insulin secretion,suggesting the involvement of glucose signaling through a phosphorylation-dependentmechanism.

Expression and subcellular localization of cysteine- and glycine-rich protein-2 in rat undifferentiated olfactory stem cells

BACKGROUND： As a member of the LIM protein family Ⅱ, cysteine- and glycine-rich protein-2 （CRP2） has been demonstrated to play a role in the regulation of growth and differentiation of eukaryotic cells. Our previous study has demonstrated that CRP2 can be detected in the embryonic rat inner ear but not in the adult rat inner ear. However, at present, the expression of LIM protein family H members in stem or precursor cells has not been described. OBJECTIVE： To determine the expression and sub-cellular localization of CRP2 in olfactory stem cells. DESIGN, TIME AND SETTING： An experiment with repeated measures was performed in the Laboratory of Otorhinolaryngology, Head and Neck Surgery, Xijing Hospital, the Fourth Military Medical University from February 2008 to April 2008. MATERIALS： Olfactory stem cells, and rabbit-anti-CRP2 polyclonal antibody were prepared and kept in our laboratory. METHODS： Reverse transcription polymerase chain reaction and Western blot analysis were used to detect expression of CRP2 in olfactory stem cells. Immunocytochemistry was also used to localize CRP2 in olfactory stem cells. MAIN OUTCOME MEASURES： The expression and sub-cellular localization of CRP2 in rat olfactory stem cells. RESULTS： CRP2 expression was found in olfactory stem cells, and CRP2 was distributed in both the nucleus and the cytoplasm. CONCLUSION： Confirmation of the expression and distribution of CRP2 in olfactory stem cells.

Multiple subcellular localizations and functions of protein kinase Cδ in liver cancer

Protein kinase Cδ(PKCδ)is a member of the PKC family,and its implications have been reported in various biological and cancerous processes,including cell proliferation,cell death,tumor suppression,and tumor progression.In liver cancer cells,accumulating reports show the bi-functional regulation of PKCδin cell death and survival.PKCδfunction is defined by various factors,such as phosphorylation,catalytic domain cleavage,and subcellular localization.PKCδhas multiple intracellular distribution patterns,ranging from the cytosol to the nucleus.We recently found a unique extracellular localization of PKCδin liver cancer and its growth factor-like function in liver cancer cells.In this review,we first discuss the structural features of PKCδand then focus on the functional diversity of PKCδbased on its subcellular localization,such as the nucleus,cell surface,and extracellular space.These findings improve our knowledge of PKCδinvolvement in the progression of liver cancer.

Molecular Determinants Responsible for the Subcellular Localization of HSV-1 UL4 Protein

The function of the herpes simplex virus type 1 （HSV-1） UL4 protein is still elusive. Our objective is to investigate the subcellular transport mechanism of the UL4 protein. In this study, fluorescence microscopy was employed to investigate the subcellular localization of UL4 and characterize the transport mechanism in living cells. By constructing a series of deletion mutants fused with enhanced yellow fluorescent protein （EYFP）, the nuclear export signals （NES） of UL4 were for the first time mapped to amino acid residues 178 to 186. In addition, the N-terminal 19 amino acids are identified to be required for the granule-like cytoplasmic pattem of UL4. Furthermore, the UL4 protein was demonstrated to be exported to the cytoplasm through the NES in a chromosomal region maintenance 1 （CRM1）-dependent manner involving RanGTP hydrolysis

Subcellular localization of Rho GTPases: implications for axon regeneration

Damage to neurons in the central nervous system often leads to a permanent loss of function due to several factors,including reduced capacity of axons to regenerate and an environment that inhibits axon regeneration because of disruption of myelin and the formation of a growth-refractory glial scar around the injury（Yoon and Tuszynski,2012）.

Analysis of Subcellular Localization and Pathogenicity of Plum Bark Necrosis Stem-Pitting Associated Virus Protein P6

Infection of plum bark necrosis stem pitting associated virus(PBNSPaV)has been reported in many Prunus species in several countries,causing significant economic losses.The very small proteins encoded by plant viruses are often overlooked due to their short sequences and uncertain significance.However,numerous studies have indicated that they might play important roles in the pathogenesis of virus infection.The role of small hydrophobic protein P6,encoded by the open reading frame 2 of PBNSPaV,has not been well explored.In this study,we amplified the P6 fragment from a PBNSPaV isolate by RT-PCR using specific primers and found that it is 174 bp long and encodes a protein of approximately 6.3 kD with a transmembrane domain.Subcellular localization analysis of P6 proteins in tobacco leaves showed that P6 localizes to the cytomembrane and nuclear membrane.To further clarify the pathogenicity of P6 proteins,we constructed a PVX-P6 expression vector by inserting the p6 fragment into a potato virus X(PVX)-based vector and transformed it into Agrobacterium tumefaciens GV3101.Infiltration of Nicotiana benthamiana(N.benthamiana)with the PVX vector-transformed A.tumefaciens led to slight mosaic symptoms at 14 days of post-inoculation.Meanwhile,infiltration with the PVX-P6 vector-transformed A.tumefaciens resulted in no significant symptoms.These results demonstrated that heterologous expression of P6 in N.benthamiana could not enhance the pathogenicity of PVX.Our study indicates that P6 may not be a potential pathogenic factor associate with the causing of symptoms,and the mode of action of PBNSPaV-P6 protein remains to be further studied.

Subcellular Localization of Small GTP-binding Protein DsRab in Dunaliella salina

With the total RNA of Dunaliella salina as a template,the cD NA sequence of D. salina small GTP-binding protein gene was amplified by RT-PCR technique,and cloned onto pM Dl8-T simple vector,the recon was subjected to PCR detection and restriction endonuclease analysis,and the total sequence of DNA was determined. The results showed that the cloned fragment was 612 bp,and shared 100% homology with reported D. salina DsRab gene（ GenB ank： JN989548）. The target gene fragment was inserted downstream of pM DCG 35 S promoter,constructing subcellular localization recombinant vector pM DCG-DsRab. The successfully constructed subcellular localization recombinant vector pM DCG-DsRab was transformed into Agrobacterium tumefaciens LBA4404,and positive single clones were screened and used for transinfection of onion epidemical cells by Agrobacterium-mediated method,and the instant expression of DsRab was observed under fluorescence microscope. The results showed that the fusion protein GFP-DsRab was successfully expressed in onion epidemical cells,and mainly distributed on cytomembrane. This study will provide reference for further illumination of the function and action mechanism of D. salina small GTP-binding protein DsRab.

SGCL-LncLoc:An Interpretable Deep Learning Model for Improving IncRNA Subcellular Localization Prediction with Supervised Graph Contrastive Learning

Understanding the subcellular localization of long non-coding RNAs(IncRNAs)is crucial for unraveling their functional mechanisms.While previous computational methods have made progress in predicting IncRNA subcellular localization,most of them ignore the sequence order information by relying on k-mer frequency features to encode IncRNA sequences.In the study,we develope SGCL-LncLoc,a novel interpretable deep learning model based on supervised graph contrastive learning.SGCL-LncLoc transforms IncRNA sequences into de Bruijn graphs and uses the Word2Vec technique to learn the node representation of the graph.Then,SGCL-LncLoc applies graph convolutional networks to learn the comprehensive graph representation.Additionally,we propose a computational method to map the attention weights of the graph nodes to the weights of nucleotides in the IncRNA sequence,allowing SGCL-LncLoc to serve as an interpretable deep learning model.Furthermore,SGCL-LncLoc employs a supervised contrastive learning strategy,which leverages the relationships between different samples and label information,guiding the model to enhance representation learning for IncRNAs.Extensive experimental results demonstrate that SGCL-LncLoc outperforms both deep learning baseline models and existing predictors,showing its capability for accurate IncRNA subcellular localization prediction.Furthermore,we conduct a motif analysis,revealing that SGCL-LncLoc successfully captures known motifs associated with IncRNA subcellular localization.The SGCL-LncLoc web server is available at http://csuligroup.com:8000/SGCL-LncLoc.The source code can be obtained from https://github.com/CSUBioGroup/SGCL-LncLoc.