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1.
Gijsbers L Man HY Kloet SK de Haan LH Keijer J Rietjens IM van der Burg B Aarts JM 《Analytical biochemistry》2011,(1):77-83
Phosphatidylinositol (PtdIns) is phosphorylated at D-3, D-4, and/or D-5 of the inositol ring to produce seven distinct lipid second messengers known as phosphoinositides (PIs). The PI level is temporally and spatially controlled at the cytosolic face of the cellular membrane. Effectors containing PI-binding domains (e.g., PH, PX, FYVE, ENTH, FERM) associate with specific PIs. This process is crucial for the localization of a variety of cell-signaling proteins, thereby regulating intracellular membrane trafficking, cell growth and survival, cytoskeletal organization, and so on. However, quantitative assessments of protein–PI interactions are generally difficult due to insolubility of PIs in aqueous solution. Here we incorporated PIs into a lipid–protein nanoscale bilayer (nanodisc), which is applied for studying the protein–PI interactions using pull-down binding assay, fluorescence polarization, and nuclear magnetic resonance studies, each facilitating fast, quantitative, and residue-specific evaluation of the protein–PI interactions. Therefore, the PI-incorporated nanodisc could be used as a versatile tool for studying the protein–lipid interactions by various biochemical and biophysical techniques. 相似文献
2.
The phosphorylated derivatives of phosphatidylinositol (PtdIns), known as the polyphosphoinositides (PIs), represent key membrane-localized signals in the regulation of fundamental cell processes, such as membrane traffic and cytoskeleton remodelling. The reversible production of the PIs is catalyzed through the combined activities of a number of specific phosphoinositide phosphatases and kinases that can either act separately or in concert on all the possible combinations of the 3, 4, and 5 positions of the inositol ring. So far, seven distinct PI species have been identified in mammalian cells and named according to their site(s) of phosphorylation: PtdIns 3-phosphate (PI3P); PtdIns 4-phosphate (PI4P); PtdIns 5-phosphate (PI5P); PtdIns 3,4-bisphosphate (PI3,4P2); PtdIns 4,5-bisphosphate (PI4,5P2); PtdIns 3,5-bisphosphate (PI3,5P2); and PtdIns 3,4,5-trisphosphate (PI3,4,5P3). Over the last decade, accumulating evidence has indicated that the different PIs serve not only as intermediates in the synthesis of the higher phosphorylated phosphoinositides, but also as regulators of different protein targets in their own right. These regulatory actions are mediated through the direct binding of their protein targets. In this way, the PIs can control the subcellular localization and activation of their various effectors, and thus execute a variety of cellular responses. To exert these functions, the metabolism of the PIs has to be finely regulated both in time and space, and this is achieved by controlling the subcellular distribution, regulation, and activation states of the enzymes involved in their synthesis and removal (kinases and phosphatases). These exist in many different isoforms, each of which appears to have a distinctive intracellular localization and regulation. As a consequence of this subcompartimentalized PI metabolism, a sort of "PI-fingerprint" of each cell membrane compartment is generated. When combined with the targeted recruitment of their protein effectors and the different intracellular distributions of other lipids and regulatory proteins (such as small GTPases), these factors can maintain and determine the identity of the cell organelles despite the extensive membrane flux []. Here, we provide an overview of the regulation and roles of different phosphoinositide kinases and phosphatases and their lipid products at the Golgi complex. 相似文献
3.
Proteasomes are multicatalytic protease complexes in the cell, involved in the non-lysosomal recycling of intra-cellular proteins. Proteasomes play a critical role in regulation of cell division in both normal as well as cancer cells. In cancer cells this homeostatic function is deregulated leading to the hyperactivation of the proteasomes. Proteasome inhibitors (PIs) are a class of compounds, which either reversibly or irreversibly block the activity of proteasomes and induce cancer cell death. Interference of PIs with the ubiquitin proteasome pathway (UPP) involved in protein turnover in the cell leads to the accumulation of proteins engaged in cell cycle progression, which ultimately put a halt to cancer cell division and induce apoptosis. Upregulation of many tumor suppressor proteins involved in cell cycle arrest are known to play a role in PI induced cell cycle arrest in a variety of cancer cells. Although many PIs target the proteasomes, not all of them are effective in cancer therapy. Some cancers develop resistance against proteasome inhibition by possibly activating compensatory signaling pathways. However, the details of the activation of these pathways and their contribution to resistance to PI therapy remain obscure. Delineation of these pathways may help in checking resistance against PIs and deducing effective combinational approaches for improved treatment strategies. This review will discuss some of the signaling pathways related to proteasome inhibition and cell division that may help explain the basis of resistance of some cancers to proteasome inhibitors and underline the need for usage of PIs in combination with traditional chemotherapy. 相似文献
4.
Phosphoinositides (PIs) undergo phosphorylation/dephosphorylation cycles through organelle-specific PI kinases and PI phosphatases that lead to distinct subcellular distributions of the individual PI species. Specific PIs control the correct timing and location of many trafficking events. Their ultimate mode of action is not always well defined, but it includes localized recruitment of transport machinery, allosteric regulation of PI-binding proteins and changes in the physical properties of the membrane. 相似文献
5.
Orawan Bunyatang Nion Chirapongsatonkul Nunta Churngchow 《Journal of plant biochemistry and biotechnology.》2013,22(2):185-192
Protease inhibitors (PIs) are one family of pathogenesis-related proteins (PR-proteins) that play essential roles in defense mechanisms against an attack by a pathogenic microorganism or insect. Cell suspension derived from a seed integument of rubber tree (Hevea brasiliensis) treated for 48 h with 20 μM copper sulphate, an abiotic elicitor, had an increased production of PIs. The intracellular PIs were detected in an extract of treated cells; however, much higher levels of PIs were found in the medium (extracellular). Using azocasein as substrate, these PIs possessed strong inhibitory activity against subtilisin A but not against trypsin, chymotrysin and papain. These extracellular PIs were purified by anion exchange chromatography, DEAE-Sepharose (CL-6B), eluted with 0.06 M NaCl in 20 mM Tris-HCl (pH 7.0). The active fractions were then subjected to native and SDS preparative gel electrophoresis, respectively. A single band of a purified PI with a molecular weight of 25 kDa was revealed after a tricine SDS-PAGE and stained with silver nitrate. The yield of this purified protein was 3.14 ng.g?1. The activity of the purified PI was stable up to 70 °C, and its activity was retained in the buffer pH values of 2–10. The biological activity of the obtained PI was investigated. It was found that the PI at 5 μg.mL?1 (0.2 μM) inhibited the mycelium growth of Phytophthora palmivora, a rubber tree pathogen. 相似文献
6.
Proteinase inhibitors (PIs) of the potato type II family have been identified in a number of solanaceous species. Most family members have two PI domains which are specific for either chymotrypsin or trypsin. More recently family members have been described with three or six repeated PI domains. Here we describe a novel four-domain family member produced in the stigmas and leaves of the ornamental tobacco, Nicotiana alata, which has high sequence identity with a six-domain member from the same species. Both proteins are produced as precursors that enter the secretory pathway and are subsequently processed into a series of 6 kDa PIs. The four- and six-domain precursor proteins were isolated from immature stigmas and characterised by mass spectrometry which revealed that both proteins had been trimmed at the N-terminus, at a position corresponding to the predicted signal peptide cleavage site. Furthermore, no post-translational modifications were apparent. 相似文献
7.
Phosphoinositides (PI) play important regulatory roles in cell physiology. Localization and quantitation of PIs within the cell is necessary to understand their precise function. Currently, ectopic expression of green fluorescent protein (GFP)-fused PI-binding domains is used to visualize PIs localized to the cell membrane. However, ectopically expressed PI-binding domains may compete with endogenous binding proteins, thus altering the physiological functions of the PIs. Here, we establish a novel method for quantification and visualization of PIs in cells and tissue samples using PI-binding domains labeled with quantum dots (Qdot) as specific probes. This method allowed us to simultaneously quantify three distinct PIs, phosphatidylinositol 3,4,5-triphosphatase [PtdIns(3,4,5)P(3)), PtdIns(3,4)P(2), and PtdIns(4,5)P(2), in crude acidic lipids extracted from insulin-stimulated cells. In addition, the method allowed the PIs to be visualized within fixed cells and tissues. Sequential and spatial changes in PI production and distribution were detected in platelet-derived growth factor (PDGF)-stimulated NRK49F cells. We also observed accumulation of PtdIns(3,4)P(2) at the dorsal ruffle in PDGF-stimulated NIH3T3 cells. Finally, we found PtdIns(3,4,5)P(3) was enriched in lung cancer tissues, which also showed high levels of phosphorylated Akt. Our new method to quantify and visualize PIs is expected to provide further insight into the role of lipid signaling in a wide range of cellular events. 相似文献
8.
β cell pseudoislets (PIs) are used for the in vitro study of β-cells in a three-dimensional (3-D) configuration. Current methods of PI induction require unique culture conditions and extensive mechanical manipulations. Here we report a novel co-culture system consisting of high passage β-cells and islet-derived endothelial cells (iECs) that results in a rapid and spontaneous formation of free-floating PIs. PI structures were formed as early as 72 h following co-culture setup and were preserved for more than 14 d. These PIs, composed solely of β-cells, were similar in size to that of native islets and showed an increased percentage of proinsulin-positive cells, increased insulin gene expression in response to glucose stimulation, and restored glucose-stimulated insulin secretion when compared to β-cells cultured as monolayers. Key extracellular matrix proteins that were absent in β-cells cultured alone were deposited by iECs on PIs and were found in and around the PIs. iEC-induced PIs are a readily available tool for examining β cell function in a native 3-D configuration and can be used for examining β-cell/iEC interactions in vitro. 相似文献
9.
Probing phosphoinositide functions in signaling and membrane trafficking 总被引:16,自引:0,他引:16
The inositol phospholipids (PIs) comprise a family of eight species with different combinations of phosphate groups arranged around the inositol ring. PIs are among the most versatile signaling molecules known, with key roles in receptor-mediated signal transduction, actin remodeling and membrane trafficking. Recent studies have identified effector proteins and specific lipid-binding domains through which PIs signal. These lipid-binding domains can be used as probes to further our understanding of the spatial and temporal control of individual PI species. New layers of complexity revealed by the use of such probes include the occurrence of PIs at intracellular locations, the identification of phosphatidylinositol signaling hotspots and the presence of non-membrane pools of PIs in cell nuclei. 相似文献
10.
Inga laurina trypsin inhibitor (ILTI) obstructs Spodoptera frugiperda trypsins expressed during adaptive mechanisms against plant protease inhibitors
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Suzy Wider Machado Caio Fernando Ramalho de Oliveira Neide Graciano Zério José Roberto Postali Parra Maria Lígia Rodrigues Macedo 《Archives of insect biochemistry and physiology》2017,95(4)
Plant protease inhibitors (PIs) are elements of a common plant defense mechanism induced in response to herbivores. The fall armyworm, Spodoptera frugiperda, a highly polyphagous lepidopteran pest, responds to various PIs in its diet by expressing genes encoding trypsins. This raises the question of whether the PI‐induced trypsins are also inhibited by other PIs, which we posed as the hypothesis that Inga laurina trypsin inhibitor (ILTI) inhibits PI‐induced trypsins in S. frugiperda. In the process of testing our hypothesis, we compared its properties with those of selected PIs, soybean Kunitz trypsin inhibitor (SKTI), Inga vera trypsin inhibitor (IVTI), Adenanthera pavonina trypsin inhibitor (ApTI), and Entada acaciifolia trypsin inhibitor (EATI). We report that ILTI is more effective in inhibiting the induced S. frugiperda trypsins than SKTI and the other PIs, which supports our hypothesis. ILTI may be more appropriate than SKTI for studies regarding adaptive mechanisms to dietary PIs. 相似文献
11.
Mariko Yokogawa Yoshihiro Kobashigawa Naoki Yoshida Kenji Ogura Kohsuke Harada Fuyuhiko Inagaki 《The Journal of biological chemistry》2012,287(42):34936-34945
Phosphoinositides (PIs) are crucial lipid components of membranes and are involved in a number of cellular processes through interactions with their effector proteins. Recently, we have established a lipid-protein nanoscale bilayer (nanodisc) containing PIs, hereafter referred to as PI-nanodisc and demonstrated that it could be used for both qualitative and quantitative evaluations of protein-membrane interactions. Here, we report further NMR analyses for obtaining structural insights at the residue-specific level between PI-binding effector protein and PI-nanodisc, using the FYVE domain of early endosome antigen 1 (EEA1), denoted as EEA1 FYVE, and PI(3)P-nanodisc as a model system. We performed a combination of the NMR analyses including chemical shift perturbation, transferred cross-saturation, and paramagnetic relaxation enhancement experiments. These enabled an identification of the interaction surface, structural change, and relative orientation of EEA1 FYVE to the PI(3)P-incorporated lipid bilayer, substantiating that NMR analyses of protein-membrane interactions using nanodisc makes it possible to show the residue-specific interactions in the lipid bilayer environment. 相似文献
12.
Phosphoinositides (PIs) are minor, but essential phospholipid constituents of eukaryotic membranes, and are involved in the regulation of various physiological processes. Recent genetic and cell biological advances indicate that PIs play important roles in the control of polar tip growth in plant cells. In root hairs and pollen tubes, PIs control directional membrane trafficking required for the delivery of cell wall material and membrane area to the growing tip. So far, the exact mechanisms by which PIs control polarity and tip growth are unresolved. However, data gained from the analysis of plant, fungal and animal systems implicate PIs in the control of cytoskeletal dynamics, ion channel activity as well as vesicle trafficking. The present review aims at giving an overview of PI roles in eukaryotic cells with a special focus on functions pertaining to the control of cell polarity. Comparative screening of plant and fungal genomes suggests diversification of the PI system with increasing organismic complexity. The evolutionary conservation of the PI system among eukaryotic cells suggests a role for PIs in tip growing cells in models where PIs so far have not been a focus of attention, such as fungal hyphae. 相似文献
13.
Houjou T Hayakawa J Watanabe R Tashima Y Maeda Y Kinoshita T Taguchi R 《Journal of lipid research》2007,48(7):1599-1606
Glycosylphosphatidylinositol (GPI) anchor is a major lipidation in posttranslational modification. GPI anchor precursors are biosynthesized from endogenous phosphatidylinositols (PIs) and attached to proteins in the endoplasmic reticulum. Endogenous PIs are characterized by domination of diacyl species and the presence of polyunsaturated fatty acyl chain, such as 18:0-20:4, at the sn-2 position. In contrast, the features of mammalian glycosylphosphatidylinositol-anchored proteins (GPI-APs) are domination of alkyl/acyl PI species and the presence of saturated fatty acyl chains at the sn-2 position, the latter being consistent with association with lipid rafts. Recent studies showed that saturated fatty acyl chain at sn-2 is introduced by fatty acid remodeling that occurs in GPI-APs. To gain insight into the former feature, we analyzed the molecular species of several different GPI precursors derived from various mammalian mutant cell lines. Here, we show that the PI species profile greatly changed in the precursor glucosamine (GlcN)-acyl-PI and became very similar to that of GPI-APs before fatty acid remodeling. They had alkyl (or alkenyl)/acyl types with unsaturated acyl chain as the major PI species. Therefore, a specific feature of the PI moieties of mature GPI-APs, domination of alkyl (or alkenyl)/acyl type species over diacyl types, is established at the stage of GlcN-acyl-PI. 相似文献
14.
Abstract: Many plant species contain chemical defenses that protect them against herbivores. Despite the benefit of these chemical defenses, not all individuals contain high levels of these compounds. In the native tobacco Nicotiana attenuata we found that plants from three natural populations differed considerably in their ability to produce trypsin protease inhibitors (PIs), which are defensive proteins that reduce herbivore damage to plants. Plants from a Utah (U) population produced high levels, whereas plants from Arizona (A) contained no detectable PI levels. Californian (C) plants had intermediate levels. The PI-producing U and C plants thus differ quantitatively from each other, whereas they both differ qualitatively from PI-deficient A plants. Here we analyze how PI production is inherited in N. attenuata with the ultimate goal of better understanding how the quantitative and qualitative differences between the three populations have evolved. Using a series of classical crossing designs, we determined that the ability to produce PIs is inherited as a dominant Mendelian trait. PI-deficient plants contain two non-functional recessive alleles, whereas heterozygous plants or homozygous dominant plants both are able to produce PIs. Similarly, the level of constitutive PIs may be determined by its genotype, either by an interaction between a functional and a non-functional allele in heterozygotes, or by a factor on the PI allele itself in homozygous C plants. Based on these data and on previous studies with A and U plants we postulate that the PI-deficient A plants may have originated from a mutant that lost its ability to produce PIs. The fitness loss due to reduced herbivore resistance may be offset by the fitness gain associated with increased competitive ability, a trade-off which may maintain this mutation in the Arizona population. 相似文献
15.
The objective of these studies was to test the hypothesis that proteins that contain potential polyisoprenyl recognition sequences (PIRSs) in their transmembrane-spanning domain can bind to the polyisoprenyl (PI) glycosyl carrier lipids undecaprenyl phosphate (C55-P) and dolichyl phosphate (C95-P). A number of prokaryotic and eukaryotic glycosyltransferases that utilize PI coenzymes contain a conserved PIRS postulated to be the active PI binding domain. To study this problem, we first determined the 3D structure of a PIRS peptide, NeuE, by homonuclear 2D 1H-nuclear magnetic resonance (NMR) spectroscopy. Experimentally generated distance constraints derived from nuclear Overhauser enhancement and torsion angle constraints derived from coupling constants were used for restrained molecular dynamics and energy minimization calculations. Molecular models of the NeuE peptide were built based on calculations of energy minimization using the DGII program NMRchitect. 3D models of dolichol (C95) and C95-P were built based on our 2D 1H-NMR nuclear Overhauser enhancement spectroscopy (NOESY) results and refined by energy minimization with respect to all atoms using the AMBER (assisted modeling with energy refinements) force field. Our energy minimization studies were carried out on a conformational model of dolichol that was originally derived from small-angle X-ray scattering and molecular mechanics methods. These results revealed that the PIs are conformationally nearly identical tripartite molecules, with their three domains arranged in a coiled, helical structure. Analyses of the intermolecular cross-peaks in the 2D NOESY spectra of PIRS peptides in the presence of PIs confirmed a highly specific interaction and identified key contact amino acids in the NeuE peptide that constituted a binding motif for interacting with the PIs. These studies also showed that subtle conformational changes occurred within both the PIs and the NeuE peptide after binding. 3D structures of the resulting molecular complexes revealed that each PI could bind more than one PIRS peptide. These studies thus represent the first evidence for a direct physical interaction between specific contact amino acids in the PIRS peptides and the PIs and supports the hypothesis of a bifunctional role for the PIs. The central idea is that these superlipids may serve as a structural scaffold to organize and stabilize in functional domains PIRS-containing proteins within multiglycosyltransferase complexes that participate in biosynthetic and translocation processes. 相似文献
16.
Praveen Krishnamoorthy Clara Sanchez-Rodriguez Ingo Heilmann Staffan Persson 《Annals of botany》2014,114(6):1049-1057
Background
Plant cell walls are complex matrices of carbohydrates and proteins that control cell morphology and provide protection and rigidity for the plant body. The construction and maintenance of this intricate system involves the delivery and recycling of its components through a precise balance of endomembrane trafficking, which is controlled by a plethora of cell signalling factors. Phosphoinositides (PIs) are one class of signalling molecules with diverse roles in vesicle trafficking and cytoskeleton structure across different kingdoms. Therefore, PIs may also play an important role in the assembly of plant cell walls.Scope
The eukaryotic PI pathway is an intricate network of different lipids, which appear to be divided in different pools that can partake in vesicle trafficking or signalling. Most of our current understanding of how PIs function in cell metabolism comes from yeast and mammalian systems; however, in recent years significant progress has been made towards a better understanding of the plant PI system. This review examines the current state of knowledge of how PIs regulate vesicle trafficking and their potential influence on plant cell-wall architecture. It considers first how PIs are formed in plants and then examines their role in the control of vesicle trafficking. Interactions between PIs and the actin cytoskeleton and small GTPases are also discussed. Future challenges for research are suggested. 相似文献17.
Protease inhibitors:recent advancement in its usage as a potential biocontrol agent for insect pest management 总被引:1,自引:0,他引:1
Sujata Singh Archana Singh Sumit Kumar Pooja Mittal Indrakant K.Singh 《Insect Science》2020,27(2):186-201
Plant derived protease inhibitors(PIs)are a promising defensin for crop im-provement and insect pest management.Although agronomist made significant efforts in utilizing PIs for managing insect pests.the potentials of PIs are still obscured.Insect ability to compensate nutrient starvation induced by dietary PI feeding using different strategies,that is,overexpression of PI-sensitive protease,expression of PI-insensitive proteases,degradation of PI,has made this innumerable collection of PIs worthless.A practical challenge for agronomist is to identify potent PI candidates,to limit insect compensatory responses and to elucidate insect compensatory and resistance mechanisms activated upon herbivory.This knowledge could be further efficiently utilized to identify potential targets for RNAi-mediated pest control.These vital genes of insects could be functionally anno-tated using the advanced gene-editing technique,CRISPR/Cas9.Contemporary research is exploiting different in silico and modern molecular biology techniques to utilize PIs in controlling insect pests efficiently.This review is structured to update recent advancements in this field,along with is chronological background. 相似文献
18.
Reproductive and storage tissues of many plants produce large amounts of serine proteinase inhibitors (PIs). The ornamental
tobacco, Nicotiana alata, produces a series of 6 kDa chymotrypsin and trypsin inhibitors that accumulate to up to 30% of soluble protein in the stigma.
These inhibitors are derived by proteolytic processing of two closely related multidomain precursor proteins. Using immunogold
electron microscopy, we find that the stigmatic PIs accumulate in both the central vacuole and in the extracellular mucilage.
Labelling with antibodies specific for the C-terminal vacuolar targeting peptide (VTS) of each precursor confirms earlier
biochemical data showing that the VTS is removed during passage through the secretory pathway. We have isolated and characterised
the extracellular population of PIs, which are largely identical to PIs isolated from whole stigmas and are functional inhibitors
of serine proteases. Subcellular fractionation of immature stigmas reveals that a sub-population of the PI precursor protein
is proteolytically processed within the endoplasmic reticulum. This proteolysis results in the removal of the vacuolar sorting
information, causing secretion of this PI population. We propose a novel mechanism whereby a single gene product may be simultaneously
trafficked to two separate compartments mediated by proteolysis early in the secretory pathway. 相似文献
19.
The generation of phosphoinositides (PIs) with spatial and temporal control is a key mechanism in cellular organization and signaling. The synthesis of PIs is mediated by PI kinases, proteins that are able to phosphorylate unique substrates at specific positions on the inositol headgroup to generate signaling molecules. Phosphatidylinositol 5 phosphate 4 kinase (PIP4K) is one such lipid kinase that is able to specifically phosphorylate phosphatidylinositol 5 phosphate, the most recently discovered PI to generate the well-known and abundant PI, phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2]. PIP4K appears to be encoded only in metazoan genomes, and several genetic studies indicate important physiological functions for these enzymes in metabolism, immune function, and growth control. PIP4K has recently been reported to localize to multiple cellular compartments, including the nucleus, plasma membrane, endosomal systems, and autophagosome. However, the biochemical activity of these enzymes that is relevant to these physiological functions remains elusive. We review recent developments in this area and highlight emerging roles for these enzymes in cellular organization. 相似文献
20.
Phosphoinositide (PI) glycerolipids are key regulators of eukaryotic signal transduction, cytoskeleton architecture and membrane dynamics. The host cell PI metabolism is targeted by intracellular bacterial pathogens, which evolved intricate strategies to modulate uptake processes and vesicle trafficking pathways. Upon entering eukaryotic host cells, pathogenic bacteria replicate in distinct vacuoles or in the host cytoplasm. Vacuolar pathogens manipulate PI levels to mimic or modify membranes of subcellular compartments and thereby establish their replicative niche. Legionella pneumophila , Brucella abortus , Mycobacterium tuberculosis and Salmonella enterica translocate effector proteins into the host cell, some of which anchor to the vacuolar membrane via PIs or enzymatically turnover PIs. Cytoplasmic pathogens target PI metabolism at the plasma membrane, thus modulating their uptake and antiapoptotic signalling pathways. Employing this strategy, Shigella flexneri directly injects a PI-modifying effector protein, while Listeria monocytogenes exploits PI metabolism indirectly by binding to transmembrane receptors. Thus, regardless of the intracellular lifestyle of the pathogen, PI metabolism is critically involved in the interactions with host cells. 相似文献