1991 — 2010 |
Menon, Anant K |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Biosynthesis of Membrane Protein Glycolipid Anchors
A variety of functionally diverse proteins are attached to the membranes of eukaryotic cells via inositol-containing glycophospholipids. One of the best studied of these proteins, the glycolipid-anchored variant surface glycoprotein (VSG) of the parasitic African trypanosome, Trypanosoma brucei, is indispensable to the survival of the parasite in its mammalian host and is an attractive target for the chemotherapeutic resolution of African sleeping sickness and animal trypanosomiasis in sub-Saharan Africa. Although a molecular definition of the glycolipid anchor is available for only a few proteins other than VSG, immunological studies show that many of the anchors are antigenically similar and therefore share common features. The structure of the glycolipids and their attachment to protein is unusual from several points of view and suggests the involvement of novel biosynthetic pathways in the assembly of the membrane anchor. The aim of this proposal is to elucidate the pathway of glycolipid anchor biosynthesis using trypanosomes as a model system. The experimental approach will exploit a cell-free system capable of synthesizing the newly identified precursor glycolipid, P2, and, an inositol-acylated form of P2 termed P3. Appropriate radiolabeled metabolites such as nucleotide sugars will be added to trypanosome membranes and lipid products will be identified by thin layer and column chromatography. Neutral glycans will be generated from purified lipids and total extracts and analyzed by a high-resolution HPLC method using the Dionex chromatography system. Using these methods, putative biosynthetic lipid intermediates and immediate donors of individual components of the glycolipid will be identified. Candidate lipid intermediates will be tested by adding back purified lipids to membranes and examining the products of continued synthesis and/or degradation. Similar experiments will be performed to determine the relationship between inositol-acylated lipids and their non-inositol- acylated counterparts. Sugar analogues and other inhibitors of glycosylation will be used to express a major surface protein with a hyperacylated glycolipid anchor, will be analyzed to examine developmentally regulated divergences in glycolipid anchor biosynthesis. These approaches will be used to arrive at a broad description of anchor assembly. Once this is available, topological probes of membrane structure will be used to examine the transmembrane synthesis and distribution of lipid intermediates. The structural similarities between the VSG anchor, the precursor glycolipids and other lipid anchors described thus far, suggest a uniform biosynthetic pathway. It is expected that an analysis of VSG glycolipid anchor synthesis will elucidate a pathway of general significance in eukaryotic cells.
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1 |
1995 |
Menon, Anant K |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Fasab Summer Conference--Lipid Modification of Proteins @ Federation of Amer Soc For Exper Biology
The objective of this proposal is to obtain partial support for a Summer Conference on 'Lipid Modifications of Proteins' to be held under the auspices of the Federation of American Societies for Experimental Biology (FASEB) from August 6 to August 11, 1995, at Copper Mountain, Colorado. The conference will be limited to about 150 scientists. There will be 9 major scientific sessions, each with 3-4 oral presentations by invited experts and speakers chosen on the basis of submitted abstracts, as well as poster sessions to encourage wider participation. Previous conferences in this area were held in 1991 and 1993 under the auspices of the American Society for Biochemistry and Molecular Biology (ASBMB); both meetings were enthusiastically received. The study of lipid-modified proteins is in a period of tremendous growth. The field has emerged from the phase of structural descriptions to one in which the enzymology and biology of these modifications are the main foci of investigation. The family of acylated and/or prenylated proteins includes the src oncogene, the Ras superfamily of small guanine nucleotide binding proteins, cell surface receptors and signal transduction molecules. In many cases the lipid modification of the protein has been shown to be critical to protein function by influencing sub-cellular location or protein-protein interactions. The more elaborate glycosylphosphatidylinositol (GPI) modification is found on a functionally diverse spectrum of mainly cell surface glycoproteins that include cell adhesion molecules important in the immune system and in development, protozoal coat proteins, and cell surface receptors. Recent work has suggested that GPI-anchored proteins may be markers of membrane structural domains that are functionally important in intracellular membrane traffic and transmembrane signalling. The biological significance of lipid-modifications of proteins is of great current interest and a conference in this area in 1995 would be useful and appropriate.
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0.909 |
1997 — 2002 |
Menon, Anant K |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Photoaffinity Labeling of Glycosyltransferases of Endoplasmic Reticulum @ University of Wisconsin Madison
proteins; enzymes; biomedical resource; biomaterials; biological products;
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1 |
2001 — 2013 |
Menon, Anant K |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Phospholipid Flip-Flop in Biogenic Membranes @ Weill Medical College of Cornell Univ
DESCRIPTION (provided by applicant): The long-term objective of this proposal is to elucidate the mechanism(s) by which polar lipids are flip-flopped across biogenic (self-synthesizing) membranes such as the endoplasmic reticulum (ER). This is an important, unresolved question in membrane biology. Discovering the molecular basis of flipping is essential to understanding how the phospholipid bilayer of biomembranes is propagated, and how the topologically complex syntheses of various glycolipids are executed. The latter processes are required for assembling biologically important cell surface molecules such as N-glycosylated and glycosylphosphatidylinositol (GPI)-anchored proteins in eukaryotes, and O-antigen-modified lipopolysaccharide in Gram-negative bacteria. (Glyco)phospholipid flip-flop does not occur at an appreciable rate in protein-free liposomes but occurs rapidly in the ER via a protein-dependent, metabolic energy-independent, bi-directional, facilitated diffusion process. We hypothesize that specific proteins, biogenic membrane flippases, facilitate the transbilayer diffusion of polar lipids in the ER, including the glycerophospholipids and isoprenoid-based glycolipids that are the focus of this proposal. The characteristics of lipid flip-flop in the ER rule out the participation of ABC transporters that have been identified as potential lipid translocators in other membrane settings. No biogenic membrane flippases have been identified that flip glycerophospholipids and isoprenoid-P-sugars, but compelling genetic evidence points to a membrane protein, Rft1p, as a flippase for dolichol-pyrophosphate based glycolipids in the ER-localized pathway of protein , N-glycosylation. We propose 2 specific aims: to characterize and identify a glycerophospholipid flippase from yeast and rat liver ER and to test biochemically the role of yeast Rft1p in flipping dolichol-PP-based glycolipids. These studies will make use of procedures that we have developed for the functional reconstitution and assay of lipid flip-flop in proteoliposomes generated from a detergent-extract of ER. We anticipate that our results will define a new class of membrane proteins for which no clear prototype currently exists and begin to address our eventual goal of obtaining a molecular definition of the mechanism of lipid flip-flop in biogenic membranes.
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1 |
2004 |
Menon, Anant K |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Faseb Conference-Protein Lipidation and Membrane Domains @ Federation of Amer Soc For Exper Biology
DESCRIPTION (provided by applicant): We request partial support for a Federation of American Biological Societies of Experimental Biology (FASEB) conference entitled 'Protein Lipidation, Signaling and Membrane Domains,' to be held July 24-29, 2004 at Tucson, AZ. This meeting will highlight exciting recent progress, and provide a needed forum for critical discussions, concerning lipid-modified proteins and membrane microdomains and their roles in processes including signal transduction, trafficking of membrane molecules and disease pathogenesis. The meeting will be organized around three main themes. First, it will provide a very timely opportunity for the exchange and critical evaluation of information about the nature of membrane microdomains in living cells and their roles in functions such as cell signaling and membrane trafficking. Recent work in these areas has generated much excitement but has also clearly indicated a need for more rigorous, multifaceted strategies to address these issues. Second, the conference will highlight recent evidence that lipid-modified proteins and membrane domains play important roles in the pathogenesis of cancer, infectious diseases and inherited disorders, and it will present current progress in the development of inhibiters of protein lipidation as potential therapeutic agents for the treatment of cancer (protein prenyltransferase inhibitors) and parasitic infections (inhibitors of enzymes involved in protein myristoylation and glycosylphosphatidylinositol [GPI-] anchorage). Finally, the meeting will feature new developments in understanding the enzymology of protein lipidation, including recent successes in characterizing the enzymes involved in coupling S-acyl and GPIanchor' groups to proteins, using combined genetic and biochemical approaches. With its unique dual focus on the complementary areas of lipid-modified proteins and membrane microdomains, this meeting will promote important sharing of information by researchers from fields ranging from biophysics and structural biology to immunology and cancer biology.
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0.909 |
2011 — 2012 |
Menon, Anant K |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Structural Analysis of the Gpi Transamidase Complex @ Weill Medical Coll of Cornell Univ
DESCRIPTION (provided by applicant): Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are ubiquitous in eukaryotes. Examples of GPI-APs include folate receptor, acetylcholinesterase, renal dipeptidase and the variant surface glycoproteins of Trypanosoma brucei, the causative agent of African sleeping sickness. Inability to synthesize GPI-APs results in embryonic lethality in mammals. Defective GPI biosynthesis in multipotent hematopoietic human stem cells causes paroxysmal nocturnal hemoglobinuria, an acquired hemolytic disease. GPI-APs are needed for fungal cell viability and they are important in diseases such as trypanosomiasis, malaria and leishmaniasis that are caused by parasitic protozoa. The GPI assembly pathway is a drug target for fungal and protozoal diseases. GPI anchoring is catalyzed by GPI transamidase (GPIT), a 5-subunit membrane- bound complex located in the endoplasmic reticulum (ER). The catalytic subunit, Gpi8, shares homology with caspases; the functional role of the other subunits is unclear, but all are required for GPIT activity. Three of the non-catalytic subunits are over-expressed in certain cancers, indicating a link between GPIT and oncogenesis. In this R21 application we propose to initiate structure-function studies of the GPIT complex using electron microscopy and X-ray crystallography. We are ultimately interested in establishing the structural organization of GPIT, delineating the role of its subunits, and understanding how this important enzyme is regulated. In two specific aims we propose to (1) analyze the endogenous GPIT complex from yeast by electron microscopy and (2) express GPIT subunits and sub-complexes for X-ray crystallographic studies. Our efforts are expected to yield a medium-resolution structure of GPIT and pave the way for a future high-resolution structure of the intact complex. These studies will have high impact as there is no structural information on GPIT; also, results obtained here will shed light on other multi-subunit membrane bound enzymes in the ER such as oligosaccharyltransferase and signal peptidase that play a critical role in processing a wide range of ER-translocated proteins, including proteins destined for GPI anchoring, but whose functional architecture remains largely a mystery.
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0.976 |
2014 — 2017 |
Accardi, Alessio [⬀] Menon, Anant K |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Ca2+-Dependent Lipid Scrambling and Ion Transport by Tmem16 Proteins @ Weill Medical Coll of Cornell Univ
DESCRIPTION (provided by applicant): Phosphatidylserine (PS) is normally sequestered in the inner leaflet of the plasma membrane and its surface exposure triggers blood clotting by activated platelets and marks apoptotic cells for phagocytic clearance. PS exposure is mediated in part by Ca2+-dependent lipid scramblases that flip lipids across the plasma membrane. Despite their importance in cell physiology, the molecular identity of the scramblases has eluded researchers for decades. Recently, TMEM16F, a member of the TMEM16 family of Ca2+-activated Cl- channels, was shown to be important for Ca2+-dependent PS exposure. Mutations in TMEM16F cause Scott syndrome, an inherited bleeding disorder associated with defective lipid scrambling in platelets. TMEM16F-null mice recapitulate this disorder, in addition to displaying other defects, such as decreased bone mineralization. Although TMEM16F is important for phospholipid scrambling in platelets, its role in the process remains unclear and controversial: it has been claimed to be a scramblase, an ion channel, or a dual function protein with both scramblase and channel activity. Our long-term goal is to elucidate the molecular bases of lipid scrambling by TMEM16F and its regulation by Ca2+. These insights will allow us to understand the etiology of Scott syndrome and the role of TMEM16F in this disease as well as in other processes. To achieve our overall goal we propose to identify the structural basis for ion and lipid transport in TMEM16 proteins, determine the function and physiological role of TMEM16F and elucidate the molecular basis of Ca2+ sensing in TMEM16 proteins. Our approach is to combine biochemical assays on purified proteins with lipid scrambling and electrophysiological measurements of the same proteins in cells. We recently succeeded in expressing, purifying and functionally reconstituting TMEM16 proteins to demonstrate their intrinsic scramblase and channel activities. We also showed that expression of TMEM16F in cells indeed leads to ion transport and lipid scrambling. Thus, we have a strong platform of preliminary data to support our approach. Our proposal to understand lipid scrambling and the physiological functions of TMEM16 proteins is highly significant, as it will identify the molecular basis of Scott syndrome and elucidate the fundamental mechanism of regulated transbilayer lipid transport, a process that is not understood in any system.
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0.976 |
2014 — 2015 |
Menon, Anant K |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Rhodopsin-Mediated Phospholipid Flipping @ Weill Medical Coll of Cornell Univ
DESCRIPTION (provided by applicant): Lipid trafficking in the retina is crucial for vision. Retinoids must move rapidly between photoreceptor cells and retinal pigment epithelial cells to regenerate the visual pigment rhodopsin after light impinges on the retina. This trafficking-dependent regeneration process is termed the visual cycle and is essential for continuous vision. Defects in lipid trafficking result in retinopathies; for example, Stargardt's macular dystrophy is caused by the inability to translocate a retinoid-phospholipid adduct across photoreceptor discs. We recently discovered a surprising new player in lipid transport within the retina: our biochemical reconstitution studies revealed that rhodopsin is an ATP-independent phospholipid translocator (flippase) capable of moving phospholipids rapidly across a membrane bilayer. This discovery provides the molecular basis for previous enigmatic observations of phospholipid flip-flop in disc membranes, and assigns a novel activity to rhodopsin in addition to its well-known function in phototransduction. Our goal in this application is to decipher the molecular mechanism by which rhodopsin flips lipids across a membrane bilayer. We propose to identify structural and dynamic features of rhodopsin's transmembrane helical bundle that are necessary for its flippase activity and also determine whether it is regulated by its membrane environment, specifically cholesterol and phospholipids with polyunsaturated acyl chains. We believe that rhodopsin's lipid flippase activity is critical for dic membrane homeostasis as it corrects the phospholipid imbalance caused by ATP-driven lipid transporters, including the Stargardt's disease transporter' ABCA4, that pump phospholipids from the lumen to the cytoplasmic face of discs. Our proposal to elucidate rhodopsin's flipping mechanism is highly significant because it will not only establish a new mechanistic paradigm in membrane transport but is also key to understanding lipid homeostasis in the retina, with implications for retinal degeneration. Mutations in rhodopsin are linked to retinitis pigmentosa, but the underlying disease-causing mechanism for many of the rhodopsin mutations is not known. Our proposed studies have the potential to reveal that some of the unexplained mutations affect flippase activity, thus clarifying aspects of this retinal disease that have remained unresolved for decades. Finally, rhodopsin is a prototypical G protein-coupled receptor (GPCR). As other GPCRs have been shown to have phospholipid flippase activity, our discoveries here will have implications beyond the visual system.
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0.976 |
2015 — 2016 |
Graumann, Johannes (co-PI) [⬀] Menon, Anant K |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Er Glycolipid Flippases and Congenital Disorders of Glycosylation @ Weill Medical Coll of Cornell Univ
? DESCRIPTION (provided by applicant): At least half the proteins encoded by the human genome are N-glycosylated, an essential post-translational modification. Defects in glycosylation underlie more than 100 human genetic disorders. For example, I-cell disease is caused by the inability to construct mannose-6-phosphate epitopes on N-glycans of lysosomal hydrolases, resulting in their secretion from cells rather than localization to lysosomes. Congenital Disorders of Glycosylation (CDGs), a family of severe inherited diseases with neurological and other symptoms, frequently result from defects in protein N-glycosylation. Major gaps remain in our understanding of basic glycosylation pathways. For example, assembly of the oligosaccharide donor for N-glycosylation requires flipping of three different glycolipids (Man5GlcNAc2-PP- dolichol (M5-DLO), mannose phosphate dolichol (MPD) and glucose phosphate dolichol (GPD)) from the cytoplasmic to the luminal side of the ER. These glycolipids have long hydrocarbon tails and very polar head groups. They represent key intermediates in the transition of the N-glycan biosynthetic pathway from the cytoplasmic to the luminal side of the ER. How they are flipped across the ER is a long-standing mystery. While there is compelling evidence that specific ER membrane proteins (flippases) are required, and that they have exquisite specificity for the lipids that they transport, the molecular identity of he flippases is not known. Our goal in this R21 application is to identify the glycolipid flippases responsible for flipping M5-DLO and MPD. We developed methods that recapitulate M5-DLO and MPD flipping in synthetic lipid vesicles and this technology now provides the cornerstone of the strategy that we will deploy to achieve our goal. We will use an innovative quantitative proteomics approach to identify flippase candidates from amongst yeast ER membrane proteins. We will then use our reconstitution-based assays to screen the candidates and evaluate their activity. This will provide conclusive evidence of function that we will corroborate by in vivo tests using yeast genetics. With this strategy we expect to identify, for the first time the flippases themselves. As our approach bypasses the limitations of traditional genetic and biochemical approaches that have thus far failed to provide the molecular identity of these flippases, we believe that we have a unique opportunity to solve this decades-old problem. Our discovery will contribute to basic cell biology by revealing a new class of transport proteins, associated with an undoubtedly novel transport mechanism, and also point to new genetic loci that are associated with CDGs.
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0.976 |
2017 — 2018 |
Khelashvili, George (co-PI) [⬀] Menon, Anant K |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Molecular Mechanism of Phospholipid Scrambling by Rhodopsin @ Weill Medical Coll of Cornell Univ
The visual pigment rhodopsin is a constitutively active lipid scramblase capable of moving phospholipids rapidly between the leaflets of a membrane bilayer. This novel activity of rhodopsin plays a key role in enabling the ABC transporter ABCA4 to prevent accumulation of retinaldehydes and mitigate the formation of Vitamin A dimers. The toxic buildup of these molecules in the retina is considered to contribute to mechanisms underlying retinopathies such as Stargardt's disease and age-related macular degeneration. Both rhodopsin and its apo-protein opsin are scramblases, and here we are interested in understanding the molecular mechanism by which they translocate phospholipids. Based on preliminary results from atomistic molecular dynamics simulations we hypothesize that lipids are transported at the interface of specific transmembrane helices in opsin. Plasma membranes lack constitutive phospholipid scramblase activity, and thereby sequester the signaling lipid phosphatidylserine (PS) in the inner leaflet until scramblases are activated. However, cells that over-express opsin unexpectedly do not display PS at their surface. We hypothesize that high cholesterol levels in the plasma membrane silence opsin's scramblase activity. We propose two specific aims to test these hypotheses via biophysical, biochemical and computational methods. In the first aim we will combine experimental and in silico analyses to test the lipid translocation pathway within opsin predicted by our molecular dynamics simulations. Using site-directed mutagenesis we will crosslink transmembrane helices and also alter the pathway environment in order to disrupt transport, as measured in our fluorescence-based scramblase assays. We will also analyze the mutant opsins by molecular dynamics simulations in order to obtain structural context and mechanistic insights into the experimental results. In the second aim, we will use cell and vesicle-based assays to determine the effect of cholesterol on lipid scrambling, in conjunction with molecular dynamics simulations of wild-type opsin in cholesterol- containing membranes. The proposed studies will provide the first insights into the mechanism of lipid scrambling. As scramblases have only recently been discovered, their molecular mechanism is unknown and the field is in its infancy. The knowledge that we gain here will set the stage for future elucidation of this process in molecular detail. This has important implications for physiology, because of the role that opsin's scramblase mechanism is likely to have in supporting the flippase activity of ABCA4 in photoreceptor disc membranes. Deficiencies in this mechanism are clearly associated with retinopathies. Moreover, because opsin's scramblase activity is shared by other G protein-coupled receptors (GPCRs) such as the ?2-adrenergic receptor, our experiments also have the potential to open a new area of GPCR biology.
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0.976 |
2017 — 2020 |
Arshavsky, Vadim Y Burns, Marie E (co-PI) [⬀] Khelashvili, George (co-PI) [⬀] Menon, Anant K |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Rhodopsin Dimerization: Mechanistic Basis and Functional Consequences @ Weill Medical Coll of Cornell Univ
The visual pigment rhodopsin is both a G protein-coupled receptor (GPCR) and a critical structural component of the outer segment of photoreceptor cells. Mutations in rhodopsin are the most common cause of autosomal dominant retinitis pigmentosa (RP), a blinding disease that afflicts more than 1.5 million individuals world- wide. Rhodopsin can activate the visual signaling cascade as a monomer, but it self-associates to form dimers and higher order multimers that have been proposed to be important for phototransduction. We discovered that certain rhodopsin mutants that cause RP through unknown mechanisms appear to be function normally as visual pigments but unlike wild type rhodopsin, they fail to dimerize when reconstituted into lipid vesicles. We hypothesize that the lack of dimerization in these mutants prevents normal photoreceptor function, leading eventually to loss of photoreceptor cells, retinal degeneration and RP pathology. The overall goal of this application is to elucidate the biological and pathobiological aspects of photoreceptor functions that rely on rhodopsin dimerization. The RP mutants we propose to analyze provide novel tools and an exciting new opportunity to study the molecular basis of rhodopsin dimerization and the specific role of dimerization in phototransduction and maintaining disc architecture. In our two specific aims, we propose to conduct a comprehensive analysis of rhodopsin dimerization from its molecular basis (and why it fails in RP mutants) to its biological role in living photoreceptors. In the first aim we will use multiscale computational approaches, combined with experimental probing and verification, to test specific hypotheses about the driving forces for rhodopsin dimerization, why dimerization is perturbed in the RP-associated rhodopsin mutants, whether heterodimerization between mutant and wildtype protein occurs and whether we can identify compensatory mutations that restore dimerization. In iterative fashion, predictions from analyses in silico will be tested in direct biochemical assays and the results will serve to refine models and computational investigations. In the second aim we will comprehensively characterize two mouse strains that homozygously express non-dimerizing rhodopsin mutants, one each corresponding to point mutations in TM1 (F45L) and TM5 (F220C) to reflect the different dimer interfaces that are directly implicated. Our goal is to conduct a ?360 degree? analysis of the heterozygous and homozygous knock-in mouse phenotypes by examining photoreceptor morphology, analyzing intracellular targeting of outer segment- resident proteins, biochemical characterization of phototransduction, and electrophysiological assessment of light responses produced by the mutant rods.
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0.976 |