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Nature Communications Jan 2022Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide...
Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide regioisomers and their low intracellular concentrations, profiling these lipids and linking a specific acyl variant to a change in biological state have been difficult. To enable the comprehensive analysis of phosphoinositide phosphorylation status and acyl chain identity, we develop PRMC-MS (Phosphoinositide Regioisomer Measurement by Chiral column chromatography and Mass Spectrometry). Using this method, we reveal a severe skewing in acyl chains in phosphoinositides in Pten-deficient prostate cancer tissues, extracellular mobilization of phosphoinositides upon expression of oncogenic PIK3CA, and a unique profile for exosomal phosphoinositides. Thus, our approach allows characterizing the dynamics of phosphoinositide acyl variants in intracellular and extracellular milieus.
Topics: Animals; Chromatography, Affinity; Class I Phosphatidylinositol 3-Kinases; Epidermal Growth Factor; Exosomes; Gene Expression; HEK293 Cells; HeLa Cells; Humans; Male; Mass Spectrometry; Metabolome; Mice; PC-3 Cells; PTEN Phosphohydrolase; Phosphatidylinositols; Prostate; Prostatic Neoplasms; Pyrimidines; Quinazolines; Stereoisomerism
PubMed: 35013169
DOI: 10.1038/s41467-021-27648-z -
The Journal of Cell Biology Sep 2023The lipid phosphatidylinositol 3,5-bisphosphate-PI(3,5)P2-is known to be a key regulator of cellular traffic in health and disease, but its cellular localization was...
The lipid phosphatidylinositol 3,5-bisphosphate-PI(3,5)P2-is known to be a key regulator of cellular traffic in health and disease, but its cellular localization was somewhat enigmatic until now, with the discovery of a new PI(3,5)P2 biosensor reported in this issue of JCB by Vines et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202209077).
Topics: Phosphatidylinositol Phosphates; Phosphatidylinositols; Biosensing Techniques
PubMed: 37578524
DOI: 10.1083/jcb.202308004 -
Current Topics in Microbiology and... 2012Diverse biological processes including cell growth and survival require transient association of proteins with cellular membranes. A large number of these proteins are... (Review)
Review
Diverse biological processes including cell growth and survival require transient association of proteins with cellular membranes. A large number of these proteins are drawn to a bilayer through binding of their modular domains to phosphoinositide (PI) lipids. Seven PI isoforms are found to concentrate in distinct pools of intracellular membranes, and this lipid compartmentalization provides an efficient way for recruiting PI-binding proteins to specific cellular organelles. The atomic-resolution structures and membrane docking mechanisms of a dozen PI effectors have been elucidated in the last decade, offering insight into the molecular basis for regulation of the PI-dependent signaling pathways. In this chapter, I summarize the mechanistic aspects of deciphering the 'PI code' by the most common PI-recognizing domains and discuss similarities and differences in the membrane anchoring mechanisms.
Topics: Animals; Cell Membrane; Humans; Phosphatidylinositols; Protein Structure, Tertiary; Signal Transduction
PubMed: 23086416
DOI: 10.1007/978-94-007-5025-8_6 -
Production and Purification of Phosphatidylinositol Mannosides from Mycobacterium smegmatis Biomass.Current Protocols Jun 2022Mycobacterium tuberculosis, the etiological agent of tuberculosis, is regarded as the most successful pathogen of humankind and a major threat to global health. The...
Mycobacterium tuberculosis, the etiological agent of tuberculosis, is regarded as the most successful pathogen of humankind and a major threat to global health. The mycobacterial cell wall is vital for cell growth, virulence, and resistance to antibiotics, and thus constitutes a unique target for drug development. To characterize the enzymes catalyzing the synthesis of the cell wall components, considerable amounts of substrates are required. Since many mycobacterial cell wall lipids, particularly phosphatidylinositol mannosides (PIMs), are not commercially available, isolation from cell biomass is the most straightforward way to obtain these compounds. In this study, we optimized a protocol to extract and purify PIM species, in particular Ac PIM and Ac PIM , which can be further used for the identification and characterization of target enzymes. PIMs were extracted from Mycobacterium smegmatis mc 155 ΔPimE using organic solvents, and purified through three consecutive chromatography steps. Thin-layer chromatography (TLC) was used in-between purification steps to evaluate the success of lipid separation, and nuclear magnetic resonance (NMR) was used for product quantification and to assess purity. Typically, from a 60 g batch of M. smegmatis biomass we were able to isolate approximately 9 mg of Ac PIM and 1.8 mg of Ac PIM . This is the first time the purification of phosphatidylinositol tetramannoside has been reported. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Growth of M. smegmatis mc 155 ∆PimE Basic Protocol 2: Extraction of lipids from M. smegmatis mc 155 ∆PimE Basic Protocol 3: Treatment of the lipid extract for isolation of phospholipids Basic Protocol 4: Isolation of phosphatidylinositol mannosides Basic Protocol 5: Quantification of phosphatidylinositol mannosides.
Topics: Biomass; Chromatography, Thin Layer; Mannosides; Mycobacterium smegmatis; Mycobacterium tuberculosis; Phosphatidylinositols
PubMed: 35758621
DOI: 10.1002/cpz1.458 -
Nature Chemical Biology Jan 2023Phosphatidylinositol 3-kinase type 2α (PI3KC2α) and related class II PI3K isoforms are of increasing biomedical interest because of their crucial roles in endocytic...
Phosphatidylinositol 3-kinase type 2α (PI3KC2α) and related class II PI3K isoforms are of increasing biomedical interest because of their crucial roles in endocytic membrane dynamics, cell division and signaling, angiogenesis, and platelet morphology and function. Herein we report the development and characterization of PhosphatidylInositol Three-kinase Class twO INhibitors (PITCOINs), potent and highly selective small-molecule inhibitors of PI3KC2α catalytic activity. PITCOIN compounds exhibit strong selectivity toward PI3KC2α due to their unique mode of interaction with the ATP-binding site of the enzyme. We demonstrate that acute inhibition of PI3KC2α-mediated synthesis of phosphatidylinositol 3-phosphates by PITCOINs impairs endocytic membrane dynamics and membrane remodeling during platelet-dependent thrombus formation. PITCOINs are potent and selective cell-permeable inhibitors of PI3KC2α function with potential biomedical applications ranging from thrombosis to diabetes and cancer.
Topics: Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Phosphatidylinositols; Phosphatidylinositol Phosphates
PubMed: 36109648
DOI: 10.1038/s41589-022-01118-z -
ELife Aug 2022The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of lipid-modifying enzymes generate the majority of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] lipids...
The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of lipid-modifying enzymes generate the majority of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] lipids found at the plasma membrane in eukaryotic cells. PI(4,5)P lipids serve a critical role in regulating receptor activation, ion channel gating, endocytosis, and actin nucleation. Here, we describe how PIP5K activity is regulated by cooperative binding to PI(4,5)P lipids and membrane-mediated dimerization of the kinase domain. In contrast to constitutively dimeric phosphatidylinositol 5-phosphate 4-kinase (PIP4K, type II PIPK), solution PIP5K exists in a weak monomer-dimer equilibrium. PIP5K monomers can associate with PI(4,5)P-containing membranes and dimerize in a protein density-dependent manner. Although dispensable for cooperative PI(4,5)P binding, dimerization enhances the catalytic efficiency of PIP5K through a mechanism consistent with allosteric regulation. Additionally, dimerization amplifies stochastic variation in the kinase reaction velocity and strengthens effects such as the recently described stochastic geometry sensing. Overall, the mechanism of PIP5K membrane binding creates a broad dynamic range of lipid kinase activities that are coupled to the density of PI(4,5)P and membrane-bound kinase.
Topics: Cell Membrane; Dimerization; Phosphates; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor)
PubMed: 35976097
DOI: 10.7554/eLife.73747 -
Biophysical Journal Dec 2021Kindlin-2, a member of the Kindlin family of peripheral membrane proteins, is important for integrin activation and stabilization of epidermal growth factor receptor. It...
Kindlin-2, a member of the Kindlin family of peripheral membrane proteins, is important for integrin activation and stabilization of epidermal growth factor receptor. It associates with the cytoplasmic face of the plasma membrane via dedicated phosphatidylinositol phosphate binding domains located in the N-terminal F0 and Pleckstrin Homology domains. These domains have binding affinity for phosphatidylinositol 4,5-bisphosphate and, to a greater degree, phosphatidylinositol 3,4,5-trisphosphate. The biological significance of the differential binding of these phosphatidylinositol phosphates to Kindlin-2 and the mechanism by which they activate Kindlin-2 are not well understood. Recently, ssNMR identified the predominant protonation states of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate near physiological pH in the presence of anionic lipids. Here, we perform atomistic simulation of the bound state of the Pleckstrin Homology and F0 domains of Kindlin-2 at membranes containing phosphatidylinositol 4,5-bisphosphate/phosphatidylinositol 3,4,5-trisphosphate with differing protonation states. This computational approach demonstrates that these two phosphatidylinositol phosphates differently modulate Kindlin-2 subdomain binding in a protonation-state-dependent manner. We speculate these variations in binding mode provide a mechanism for intracellular pH and Ca influx to control the membrane binding behavior and activity of Kindlin-2.
Topics: Cell Membrane; Phosphatidylinositol Phosphates; Phosphatidylinositols; Protein Binding; Protein Structure, Tertiary
PubMed: 34813727
DOI: 10.1016/j.bpj.2021.11.021 -
Biochimica Et Biophysica Acta.... Feb 2020Epithelial cells are polarized cells and typically display distinct plasma membrane domains: basal plasma membrane domains face the underlying tissue, lateral domains... (Review)
Review
Epithelial cells are polarized cells and typically display distinct plasma membrane domains: basal plasma membrane domains face the underlying tissue, lateral domains contact adjacent cells and apical domains face the exterior lumen. Each membrane domain is endowed with a specific macromolecular composition that constitutes the functional identity of that domain. Defects in apical-basal plasma membrane polarity altogether or more subtle defects in the composition of either apical or basal plasma membrane domain can give rise to severe diseases. Lipids are the main component of cellular membranes and mechanisms that control their polarized distribution in epithelial cells are emerging. In particular sphingolipids and phosphatidylinositol lipids have taken center stage in the organization of the apical and basolateral plasma membrane domain. This short review article discusses mechanisms that contribute to the polarized distribution of lipids in epithelial cells.
Topics: Animals; Cell Polarity; Endosomes; Epithelial Cells; Humans; Phosphatidylinositols
PubMed: 31809710
DOI: 10.1016/j.bbamem.2019.183145 -
Journal of Lipid Research Nov 2016The diverse family of inositol lipids is now known to be central to many aspects of cell biology. The route from the first discovery of inositol to our present day... (Review)
Review
The diverse family of inositol lipids is now known to be central to many aspects of cell biology. The route from the first discovery of inositol to our present day knowledge of inositol lipids spans more than 150 years and is long and complex. This is a brief account of some of the most important stages along that route.
Topics: Animals; Humans; Inositol; Phosphatidylinositols; Phospholipids; Type C Phospholipases
PubMed: 27623846
DOI: 10.1194/jlr.R071712 -
Progress in Lipid Research Jul 2010Phosphoinositides are essential signaling molecules linked to a diverse array of cellular processes in eukaryotic cells. The metabolic interconversions of these... (Review)
Review
Phosphoinositides are essential signaling molecules linked to a diverse array of cellular processes in eukaryotic cells. The metabolic interconversions of these phospholipids are subject to exquisite spatial and temporal regulation executed by arrays of phosphatidylinositol (PtdIns) and phosphoinositide-metabolizing enzymes. These include PtdIns- and phosphoinositide-kinases that drive phosphoinositide synthesis, and phospholipases and phosphatases that regulate phosphoinositide degradation. In the past decade, phosphoinositide phosphatases have emerged as topics of particular interest. This interest is driven by the recent appreciation that these enzymes represent primary mechanisms for phosphoinositide degradation, and because of their ever-increasing connections with human diseases. Herein, we review the biochemical properties of six major phosphoinositide phosphatases, the functional involvements of these enzymes in regulating phosphoinositide metabolism, the pathologies that arise from functional derangements of individual phosphatases, and recent ideas concerning the involvements of phosphoinositide phosphatases in membrane traffic control.
Topics: Biological Transport; Carrier Proteins; Disease; Humans; Inositol Polyphosphate 5-Phosphatases; Membrane Proteins; Nerve Tissue Proteins; PTEN Phosphohydrolase; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Protein Tyrosine Phosphatases, Non-Receptor
PubMed: 20043944
DOI: 10.1016/j.plipres.2009.12.001