SherLoc: high-accuracy prediction of protein subcellular localization by integrating text and protein sequence data

MOTIVATION: Knowing the localization of a protein within the cell helps elucidate its role in biological processes, its function and its potential as a drug target. Thus, subcellular localization prediction is an active research area. Numerous localization prediction systems are described in the literature; some focus on specific localizations or organisms, while others attempt to cover a wide range of localizations. RESULTS: We introduce SherLoc, a new comprehensive system for predicting the localization of eukaryotic proteins. It integrates several types of sequence and text-based features. While applying the widely used support vector machines (SVMs), SherLoc's main novelty lies in the way in which it selects its text sources and features, and integrates those with sequence-based features. We test SherLoc on previously used datasets, as well as on a new set devised specifically to test its predictive power, and show that SherLoc consistently improves on previous reported results. We also report the results of applying SherLoc to a large set of yet-unlocalized proteins. AVAILABILITY: SherLoc, along with Supplementary Information, is available at: http://www-bs.informatik.uni-tuebingen.de/Services/SherLoc/     

Wounding by bombardment yields highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.)

Highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.) was obtained by first wounding stem explants via microprojectile bombardment. When this was followed by cocultivation with disarmed Agrobacterium in the dark, the transformation frequency-based on transient GUS expression-increased to over 10-fold that of explants wounded by other means and cocultivated under constant light. Two cycles of regeneration/selection on kanamycin were employed to generate stably transformed carnation plants and eliminate chimeras: first, plantlets were regenerated from inoculated stem explants and then leaves from these plantlets were used to generate transgenes in a second selection cycle of adventitious shoot regeneration. Agrobacterium strain AGLO, carrying the binary vector pCGN7001 containing uidA and nptII genes, was used in the stable transformation experiments. The combination of wounding via bombardment, cocultivation in the dark and two cycles of kanamycin selection yielded an overall transformation efficiency of 1–2 transgenes per 10 stem explants for the three carnation varieties analyzed. Histochemical and molecular analyses of marker genes in T₀ and T₁ generations confirmed the transgenic nature of the selected plants.     

Improving the analytical toolbox to investigate copurifying host cell proteins presence: N-(4)-(β-acetylglucosaminyl)-l-asparaginase case study

Levels of host cell proteins (HCPs) in purification intermediates and drug substances (DS) of monoclonal antibodies (mAbs) must be carefully monitored for the production of safe and efficacious biotherapeutics. During the development of mAb1, an immunoglobulin G1 product, unexpected results generated with HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit triggered an investigation which led to the identification of a copurifying HCP called N-(4)-(β-acetylglucosaminyl)-l -asparaginase (AGA, EC3.5.1.26) by liquid chromatography–tandem mass spectrometry (LC-MS/MS). The risk assessment performed indicated a low immunogenicity risk for the copurifying HCP and an ad hoc stability study demonstrated no mAb glycan cleavage and thus no impact on product quality. Fractionation studies performed on polishing steps revealed that AGA was coeluted with the mAb. Very interestingly, the native digestion protocol implemented to go deeper in the MS–HCP profiling was found to be incompatible with correct AGA detection in last purification intermediate and DS, further suggesting a hitchhiking behavior of AGA. In silico surface characterization of AGA also supports this hypothesis. Finally, the combined support of HCP ELISA results and MS allowed process optimization and removal of this copurifying HCP.     

Reduced KIR2DL1 recognition of MHC class I molecules presenting phosphorylated peptides

As initially described by K. Karre and colleagues in the missing self hypothesis, cells expressing self-MHC class I proteins are protected from NK cells attack. In contrast, reduction in the expression of MHC class I molecules due to viral infection or tumor transformation result in the killing of these "abnormal" cells by NK cells via NK-activating receptors. Thus, NK killing of target cells is determined by both negative signals coming from MHC class I proteins and by positive signals derived from the activating ligands. The bound peptide in MHC class I play an important role in the balanced recognition of NK cells. The peptide stabilizes the MHC complex and interacts directly with the NK inhibitory receptors, thus participating in the determination of the fate of the target cells. In this study we demonstrate that posttranslational modifications such as phosphorylation of the presented peptide altered the ability of NK cells to recognize MHC class I molecules. By using a consensus peptide (QYDDAVYKL) that binds HLA-Cw4 in which different positions in the bound peptide were modified by serine phosphorylation, we observed a reduction in KIR2DL1 binding that led to decreased protection from NK killing. Therefore, it might be possible that alteration in the phosphorylation pattern during tumor transformation or viral infection may result in less inhibition and, consequently, improved NK cell killing.     

Sequential phosphorylation of insulin receptor substrate-2 by glycogen synthase kinase-3 and c-Jun NH2-terminal kinase plays a role in hepatic insulin signaling

Serine phosphorylation of insulin receptor substrate (IRS) proteins is a potential inhibitory mechanism in insulin signaling. Here we show that IRS-2 is phosphorylated by glycogen synthase kinase (GSK)-3. Phosphorylation by GSK-3 requires prior phosphorylation of its substrates, prompting us to identify the "priming kinase." It was found that the stress activator anisomycin enhanced the ability of GSK-3 to phosphorylate IRS-2. Use of a selective c-Jun NH(2)-terminal kinase (JNK) inhibitor and cells overexpressing JNK implicated JNK as the priming kinase. This allowed us to narrow down the number of potential GSK-3 phosphorylation sites within IRS-2 to four regions that follow the motif SXXXSP. IRS-2 deletion mutants enabled us to localize the GSK-3 and JNK phosphorylation sites to serines 484 and 488, respectively. Mutation at serine 488 reduced JNK phosphorylation of IRS-2, and mutation of each site separately abolished GSK-3 phosphorylation of IRS-2. Treatment of H4IIE liver cells with anisomycin inhibited insulin-induced tyrosine phosphorylation of IRS-2; inhibition was reversed by pretreatment with the JNK and GSK-3 inhibitors. Moreover, overexpression of JNK and GSK-3 in H4IIE cells reduced insulin-induced tyrosine phosphorylation of IRS-2 and its association with the p85 regulatory subunit of phosphatidylinositol 3-kinase. Finally, both GSK-3 and JNK are abnormally upregulated in the diabetic livers of ob/ob mice. Together, our data indicate that IRS-2 is sequentially phosphorylated by JNK and GSK-3 at serines 484/488 and provide evidence for their inhibitory role in hepatic insulin signaling.     

Multi-dimensional classification of biomedical text: toward automated, practical provision of high-utility text to diverse users

MOTIVATION: Much current research in biomedical text mining is concerned with serving biologists by extracting certain information from scientific text. We note that there is no 'average biologist' client; different users have distinct needs. For instance, as noted in past evaluation efforts (BioCreative, TREC, KDD) database curators are often interested in sentences showing experimental evidence and methods. Conversely, lab scientists searching for known information about a protein may seek facts, typically stated with high confidence. Text-mining systems can target specific end-users and become more effective, if the system can first identify text regions rich in the type of scientific content that is of interest to the user, retrieve documents that have many such regions, and focus on fact extraction from these regions. Here, we study the ability to characterize and classify such text automatically. We have recently introduced a multi-dimensional categorization and annotation scheme, developed to be applicable to a wide variety of biomedical documents and scientific statements, while intended to support specific biomedical retrieval and extraction tasks. RESULTS: The annotation scheme was applied to a large corpus in a controlled effort by eight independent annotators, where three individual annotators independently tagged each sentence. We then trained and tested machine learning classifiers to automatically categorize sentence fragments based on the annotation. We discuss here the issues involved in this task, and present an overview of the results. The latter strongly suggest that automatic annotation along most of the dimensions is highly feasible, and that this new framework for scientific sentence categorization is applicable in practice.     

In Planta Production of Indole-3-Acetic Acid by Colletotrichum gloeosporioides f. sp. aeschynomene

The plant pathogenic fungus Colletotrichum gloeosporioides f. sp. aeschynomene utilizes external tryptophan to produce indole-3-acetic acid (IAA) through the intermediate indole-3-acetamide (IAM). We studied the effects of tryptophan, IAA, and IAM on IAA biosynthesis in fungal axenic cultures and on in planta IAA production by the fungus. IAA biosynthesis was strictly dependent on external tryptophan and was enhanced by tryptophan and IAM. The fungus produced IAM and IAA in planta during the biotrophic and necrotrophic phases of infection. The amounts of IAA produced per fungal biomass were highest during the biotrophic phase. IAA production by this plant pathogen might be important during early stages of plant colonization.     

Light affects the accessibility of the thylakoid light harvesting complex II (LHCII) phosphorylation site to the membrane protein kinase(s)

Redox-controlled, reversible phosphorylation of the thylakoid light harvesting complex II (LHCII) regulates its association with photosystems (PS) I or II and thus, energy distribution between the two photosystems (state transition). Illumination of solubilized LHCII enhances exposure of the phosphorylation site at its N-terminal domain to protein kinase(s) and tryptic cleavage in vitro [Zer et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282]. Here we report that short illumination (5-10 min, 15-30 micromol m(-2) s(-1)) enhances the accessibility of LHCII phosphorylation site to kinase(s) activity also in isolated thylakoids. However, prolonged illumination or higher light intensities (30 min, 80-800 micromol m(-2) s(-1)) prevent phosphorylation of LHCII in the isolated membranes as well as in vivo, although redox-dependent protein kinase activity persists in the illuminated thylakoids toward exogenous solubilized LHCII. This phenomenon, ascribed to light-induced inaccessibility of the phosphorylation site to the protein kinase(s), affects in a similar way the accessibility of thylakoid LHCII N-terminal domain to tryptic cleavage. The illumination effect is not redox related, decreases linearly with temperature from 25 to 5 degrees C and may be ascribed to light-induced conformational changes in the complex causing lateral aggregation of dephosphorylated LHCII bound to and/or dissociated from PSII. The later state occurs under conditions allowing turnover of the phospho-LHCII phosphate. The light-induced inaccessibility of LHCII to the membrane-bound protein kinase reverses readily in darkness only if induced under LHCII-phosphate turnover conditions. Thus, phosphorylation prevents irreversible light-induced conformational changes in LHCII allowing lateral migration of the complex and the related state transition process.     

Eukaryotic RNase P: role of RNA and protein subunits of a primordial catalytic ribonucleoprotein in RNA-based catalysis

Ribonuclease P (RNase P) is an essential enzyme that processes the 5' leader sequence of precursor tRNA. Eubacterial RNase P is an RNA enzyme, while its eukaryotic counterpart acts as catalytic ribonucleoprotein, consisting of RNA and numerous protein subunits. To study the latter form, we reconstitute human RNase P activity, demonstrating that the subunits H1 RNA, Rpp21, and Rpp29 are sufficient for 5' cleavage of precursor tRNA. The reconstituted RNase P precisely delineates its cleavage sites in various substrates and hydrolyzes the phosphodiester bond. Rpp21 and Rpp29 facilitate catalysis by H1 RNA, which seems to require a phylogenetically conserved pseudoknot structure for function. Unexpectedly, Rpp29 forms a catalytic complex with M1 RNA of E. coli RNase P. The results uncover the core components of eukaryotic RNase P, reveal its evolutionary origin in translation, and provide a paradigm for studying RNA-based catalysis by other nuclear and nucleolar ribonucleoprotein enzymes.