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The Biochemical Journal Mar 2004All mycobacterial species, including pathogenic Mycobacterium tuberculosis, synthesize an abundant class of phosphatidylinositol mannosides (PIMs) that are essential for...
All mycobacterial species, including pathogenic Mycobacterium tuberculosis, synthesize an abundant class of phosphatidylinositol mannosides (PIMs) that are essential for normal growth and viability. These glycolipids are important cell-wall and/or plasma-membrane components in their own right and can also be hyperglycosylated to form other wall components, such as lipomannan and lipoarabinomannan. We have investigated the steps involved in the biosynthesis of the major PIM species in a new M. smegmatis cell-free system. A number of apolar and polar PIM intermediates were labelled when this system was continuously labelled or pulse-chase-labelled with GDP-[3H]Man, and the glycan head groups and the acylation states of these species were determined by chemical and enzymic treatments and octyl-Sepharose chromatography respectively. These analyses showed that (1) the major apolar PIM species, acyl-PIM2, can be synthesized by at least two pathways that differ in the timing of the first acylation step, (2) early PIM intermediates containing a single mannose residue can be modified with two fatty acid residues, (3) formation of polar PIM species from acyl-PIM2 is amphomycin-sensitive, indicating that polyprenol phosphate-Man, rather than GDP-Man, is the donor for these reactions, (4) modification of acylated PIM4 with alpha1-2- or alpha1-6-linked mannose residues is probably the branch point in the biosyntheses of polar PIM and lipoarabinomannan respectively and (5) GDP strongly inhibits the synthesis of early PIM intermediates and increases the turnover of polyprenol phosphate-Man. These findings are incorporated into a revised pathway for mycobacterial PIM biosynthesis.
Topics: Carbohydrate Sequence; Cell Wall; Guanosine Diphosphate; Models, Chemical; Molecular Sequence Data; Mycobacterium smegmatis; Phosphatidylinositols; Polyisoprenyl Phosphate Monosaccharides
PubMed: 14627436
DOI: 10.1042/BJ20031372 -
Journal of Separation Science Aug 2023The phosphatidylinositols and phosphatidylinositol phosphates are a set of closely related lipids known to influence various cellular functions. Irregular distributions...
The phosphatidylinositols and phosphatidylinositol phosphates are a set of closely related lipids known to influence various cellular functions. Irregular distributions of these molecules have been correlated with the development and progression of multiple diseases, including Alzheimer's, bipolar disorder, and various cancers. As a result, there is continued interest regarding the speciation of these compounds, with specific consideration on how their distribution may differ between healthy and diseased tissue. The comprehensive analysis of these compounds is challenging due to their varied and unique chemical characteristics, and current generalized lipidomics methods have proven unsuitable for phosphatidylinositol analysis and remain incapable of phosphatidylinositol phosphate analysis. Here we improved upon current methods by enabling the sensitive and simultaneous analysis of phosphatidylinositol and phosphatidylinositol phosphate species, whilst enhancing their characterization through chromatographic resolution between isomeric species. A 1 mM ammonium bicarbonate and ammonia buffer was determined optimal for this goal, enabling the identification of 148 phosphatidylinositide species, including 23 lyso-phosphatidylinositols, 51 phosphatidylinositols, 59 oxidized-phosphatidylinositols, and 15 phosphatidylinositol phosphates. As a result of this analysis, four distinct canola cultivars were differentiated based exclusively on their unique phosphatidylinositide-lipidome, indicating analyses of this type may be of use when considering the development and progression of the disease through lipidomic profiles.
Topics: Phosphatidylinositols; Phosphatidylinositol Phosphates; Chromatography; Phosphates
PubMed: 37329204
DOI: 10.1002/jssc.202300165 -
The Journal of Physical Chemistry. B Aug 2021The binding of phosphatidylinositol 4,5-bisphosphate (PIP2) to the ion channel transient receptor potential vanilloid 5 (TRPV5) is critical for its function. We use...
Impact of the Protonation State of Phosphatidylinositol 4,5-Bisphosphate (PIP2) on the Binding Kinetics and Thermodynamics to Transient Receptor Potential Vanilloid (TRPV5): A Milestoning Study.
The binding of phosphatidylinositol 4,5-bisphosphate (PIP2) to the ion channel transient receptor potential vanilloid 5 (TRPV5) is critical for its function. We use atomically detailed simulations and the milestoning theory to compute the free energy profile and the kinetics of PIP2 binding to TRPV5. We estimate the rate of binding and the impact of the protonation state on the process. Several channel residues are identified as influential in the association event and will be interesting targets for mutation analysis. Our simulations reveal that PIP2 binds to TRPV5 in an unprotonated state and is protonated in the membrane. The switch between the protonation state of PIP2 is modeled as a diabatic transition and occurs about halfway through the reaction.
Topics: Biophysical Phenomena; Kinetics; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Thermodynamics
PubMed: 34396776
DOI: 10.1021/acs.jpcb.1c04052 -
Nature Sep 1992Inositol phospholipid turnover is enhanced during mitogenic stimulation of cells by growth factors and the breakdown of phosphatidylinositol 4,5-bisphosphate (PtdInsP2)...
Inositol phospholipid turnover is enhanced during mitogenic stimulation of cells by growth factors and the breakdown of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) may be important in triggering cell proliferation. PtdInsP2 also binds actin-binding proteins to regulate their activity, but it is not yet understood how this control is achieved. The protein alpha-actinin from striated muscle contains large amounts of endogenous PtdInsP2, whereas that from smooth muscle has only a little but will bind exogenously added PtdInsP2. In vitro alpha-actinin binds to F-actin and will crosslink actin filaments, increasing the viscosity of F-actin solutions. We report here that alpha-actinin from striated muscle is an endogenous PtdInsP2-bound protein and that the specific interaction between alpha-actinin and PtdInsP2 regulates the F-actin-gelating activity of alpha-actinin. Although the F-actin-gelating activity of alpha-actinin from smooth muscle is much reduced compared with that from striated muscle, exogenous PtdInsP2 can enhance the activity of smooth muscle alpha-actinin to the level seen in striated muscles. These results show that PtdInsP2 is present in striated muscle alpha-actinin and that it is necessary for alpha-actinin to realize its maximum gelating activity.
Topics: Actinin; Actins; Animals; Blotting, Western; Chickens; Dose-Response Relationship, Drug; Gene Expression Regulation; In Vitro Techniques; Muscles; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylinositols; Viscosity
PubMed: 1326084
DOI: 10.1038/359150a0 -
Proteins Oct 2021The 18.5-kDa isoform of myelin basic protein (MBP) interacts with the membrane surface of the myelin sheath to construct its compact multilamellar structure. This study...
Conformation of myelin basic protein bound to phosphatidylinositol membrane characterized by vacuum-ultraviolet circular-dichroism spectroscopy and molecular-dynamics simulations.
The 18.5-kDa isoform of myelin basic protein (MBP) interacts with the membrane surface of the myelin sheath to construct its compact multilamellar structure. This study characterized the conformation of MBP in the membrane by measuring the vacuum-ultraviolet circular-dichroism (VUVCD) spectra of MBP in the bilayer liposome comprising the following essential lipid constituents of the myelin sheath: phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2). The spectra of MBP exhibited the characteristic peaks of the helix structure in the presence of PI liposome, and the intensity increased markedly in the presence of PIP and PIP2 liposomes to show an isodichroic point. This suggests that the amount of the membrane-bound conformation of MBP enhanced due to the increased number of negative net charges on the liposome surfaces. Secondary-structure analysis revealed that MBP in the membrane comprised approximately 40% helix contents and eight helix segments. Molecular-dynamics (MD) simulations of the eight segments were conducted for 250 ns in the presence of PI membrane, which predicted two amphiphilic and three nonamphiphilic helices as the membrane-interaction sites. Further analysis of the distances of the amino-acid residues in each segment from the phosphate group suggested that the nonamphiphilic helices interact with the membrane surface electrostatically, while the amphiphilic ones invade the inside of the membrane to produce electrostatic and hydrophobic interactions. These results show that MBP can interact with the PI membrane via amphiphilic and nonamphiphilic helices under the control of a delicate balance between electrostatic and hydrophobic interactions.
Topics: Animals; Cattle; Liposomes; Myelin Basic Protein; Phosphatidylinositols; Protein Structure, Secondary
PubMed: 33998060
DOI: 10.1002/prot.26146 -
Acta Crystallographica. Section D,... Jul 2022Sec14-like phosphatidylinositol transfer proteins (PITPs) are involved in lipid metabolism and phosphatidylinositol 4-phosphate signaling by transporting...
Sec14-like phosphatidylinositol transfer proteins (PITPs) are involved in lipid metabolism and phosphatidylinositol 4-phosphate signaling by transporting phosphatidylinositol (PI) and a secondary ligand between the organellar membranes in eukaryotes. Yeast Sfh2 is a PITP that transfers PI and squalene without phosphatidylcholine transfer activity. To investigate the structural determinants for ligand specificity and transport in Sfh2, crystal structures of Sfh2 in complex with PI and squalene were determined at 1.5 and 2.4 Å resolution, respectively. The inositol head group of PI is recognized by highly conserved residues around the pocket entrance. The acyl chains of PI bind into a large hydrophobic cavity. Squalene is accommodated in the bottom of the cavity entirely by hydrophobic interactions. The binding of PI and squalene are mutually exclusive due to their overlapping binding sites, correlating with the role in lipid exchange. The binding mode of PI is well conserved in Sfh family proteins. However, squalene binding is unique to the Sfh2 homolog due to the specific hydrophobic residues forming a shape-complementary binding pocket. Recombinant apo Sfh2 forms a homodimer in vitro by the hydrophobic interaction of the gating α10-α11 helices in an open conformation. Ligand binding closes the lid and dissociates the dimer into monomers. This study reveals the structural determinants for the recognition of the conserved PI and a secondary ligand, squalene, and provides implications for the lipid-transfer function of Sfh2.
Topics: Ligands; Phosphatidylinositols; Phospholipid Transfer Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Squalene
PubMed: 35775985
DOI: 10.1107/S2059798322005666 -
Organic & Biomolecular Chemistry May 2020AcPIM is a potential biosynthetic intermediate for phosphatidylinositol mannosides (PIMs) from Mycobacterium tuberculosis. We achieved the first synthesis of AcPIM by...
AcPIM is a potential biosynthetic intermediate for phosphatidylinositol mannosides (PIMs) from Mycobacterium tuberculosis. We achieved the first synthesis of AcPIM by utilizing an allyl-type protecting group strategy and regioselective phosphorylation of inositol. A very potent agonist of an innate immune receptor DCAR, which is better than previously known agonists, is demonstrated.
Topics: Animals; Cytokines; Immunomodulation; Lectins, C-Type; Mice; Mycobacterium tuberculosis; Phosphatidylinositols; Phosphorylation; RAW 264.7 Cells; Receptors, Immunologic
PubMed: 32356529
DOI: 10.1039/c9ob02724f -
Biological & Pharmaceutical Bulletin Sep 2007Phosphoinositides are a family of phosphorylated derivatives of the membrane lipid phosphatidylinositol. These lipids are highly concentrated in distinct pools located... (Review)
Review
Phosphoinositides are a family of phosphorylated derivatives of the membrane lipid phosphatidylinositol. These lipids are highly concentrated in distinct pools located in a cell's plasma membrane, endosomes or nucleus, where they function as ligands for phosphoinositide-binding proteins. Protein domains that bind phosphoinositides include the pleckstrin homology (PH) domain, the phox homology (PX) domain and the Fab1p-YOPB-Vps27p-EEA1 (FYVE) domain. These domains are found in many proteins involved in intracellular signaling, membrane trafficking and cytoskeletal rearrangement. Recent studies have identified potential links between alterations to various signaling pathways involving phosphoinositides and the etiology of many human diseases.
Topics: Animals; Blood Proteins; Humans; Phosphatidylinositols; Phosphoproteins; Signal Transduction
PubMed: 17827706
DOI: 10.1248/bpb.30.1599 -
Advances in Enzyme Regulation 2011
Review
Topics: Animals; Cell Nucleus; Humans; Phosphatidylinositol Phosphates; Phosphatidylinositols; Signal Transduction
PubMed: 21035491
DOI: 10.1016/j.advenzreg.2010.09.009 -
Chemical Communications (Cambridge,... Nov 2020Phosphatidylinositol mannosides (PIMs) have been investigated as lipidic antigens for a new subunit tuberculosis vaccine. A non-natural diacylated phosphatidylinositol...
Phosphatidylinositol mannosides (PIMs) have been investigated as lipidic antigens for a new subunit tuberculosis vaccine. A non-natural diacylated phosphatidylinositol mannoside (Ac2PIM2) was designed and synthesized by mimicking the natural PIM6 processing procedure in dentritic cells. This synthetic Ac2PIM2 was achieved from α-methyl d-glucopyranoside 1 in 17 steps in 2.5% overall yield. A key feature of the strategy was extending the use of the chiral myo-inositol building block A to the O-2 and O-6 positions of the inositol unit to allow for introducing the mannose building blocks B1 and B2, and to the O-1 position for the phosphoglycerol building block C. Building block A, being a flexible core unit, may facilitate future access to other higher-order PIM analogues. A preliminary antigenic study showed that the synthetic PIM epitope (Ac2PIM2) was significantly more active than natural Ac2PIM2, which indicated that the synthetic Ac2PIM2 can be strongly immunoactive and may be developed as a potential vaccine.
Topics: Antigen-Antibody Reactions; Antigens; Carbohydrate Conformation; Epitopes; Mycobacterium tuberculosis; Phosphatidylinositols
PubMed: 33104149
DOI: 10.1039/d0cc05573e