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Lipid rafts are dynamic, nanoscale assemblies of specific proteins and lipids, distributed heterogeneously on eukaryotic membrane. Flotillin-1, a conserved eukaryotic raft marker protein (RMP) harbor SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) and oligomerization domains to regulate various cellular processes through its interactions with other signaling or transport proteins. Rafts were thought to be absent in prokaryotes hitherto, but recent report of its presence and significance in physiology of Bacillus subtilis prompted us to investigate the same in pathogenic bacteria (PB) also. In prokaryotes, proteins of SPFH2a subfamily show highest identity to SPFH domain of Flotillin-1. Moreover, bacterial genome organization revealed that Flotillin homologue harbouring SPFH2a domain exists in an operon with an upstream gene containing NFeD domain. Here, presence of RMP in PB was initially investigated in silico by analyzing the presence of SPFH2a, oligomerization domains in the concerned gene and NfeD domain in the adjacent upstream gene. After investigating 300 PB, 4 were found to harbor RMP. Among them, domains of Bas0525 (FlotP) of Bacillus anthracis (BA) showed highest identity with characteristic domains of RMP. Considering the global threat of BA as the bioterror agent, it was selected as a model for further in vitro characterization of rafts in PB. In silico and in vitro analysis showed significant similarity of FlotP with numerous attributes of Flotillin-1. Its punctate distribution on membrane with exclusive localization in detergent resistant membrane fraction; strongly favors presence of raft with RMP FlotP in BA. Furthermore, significant effect of Zaragozic acid (ZA), a raft associated lipid biosynthesis inhibitor, on several patho-physiological attributes of BA such as growth, morphology, membrane rigidity etc., were also observed. Specifically, a considerable decrease in membrane rigidity, strongly recommended presence of an unknown raft associated lipid molecule on membrane of BA. In addition, treatment with ZA decreased secretion of anthrax toxins and FlotP expression, suggesting potential role of raft in pathogenesis and physiology of BA. Thus, the present study not only suggest the existence and role of raft like entity in pathophysiology of BA but also its possible use for the development of novel drugs or vaccines against anthrax.

Gangliosides are expressed in the outer leaflet of the plasma membrane of the cells of all vertebrates and are particularly abundant in the nervous system. Ganglioside metabolism is closely associated with the pathology of Alzheimer's disease (AD). AD, the most common form of dementia, is a progressive degenerative disease of the brain characterized clinically by progressive loss of memory and cognitive function and eventually death. Neuropathologically, AD is characterized by amyloid deposits or “senile plaques,” which consist mainly of aggregated variants of amyloid β-protein (Aβ). Aβ undergoes a conformational transition from random coil to ordered structure rich in β-sheets, especially after addition of lipid vesicles containing GM1 ganglioside. In AD brain, a complex of GM1 and Aβ, termed “GAβ,” has been found to accumulate. In recent years, Aβ and GM1 have been identified in microdomains or lipid rafts. The functional roles of these microdomains in cellular processes are now beginning to unfold. Several articles also have documented the involvement of these microdomains in the pathogenesis of certain neurodegenerative diseases, such as AD. A pivotal neuroprotective role of gangliosides has been reported in in vivo and in vitro models of neuronal injury, Parkinsonism, and related diseases. Here we describe the possible involvement of gangliosides in the development of AD and the therapeutic potentials of gangliosides in this disorder.

Lipid rafts are dynamic, nanoscale assemblies of specific proteins and lipids, distributed heterogeneously on eukaryotic membrane. Flotillin-1, a conserved eukaryotic raft marker protein (RMP) harbor SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) and oligomerization domains to regulate various cellular processes through its interactions with other signaling or transport proteins. Rafts were thought to be absent in prokaryotes hitherto, but recent report of its presence and significance in physiology of Bacillus subtilis prompted us to investigate the same in pathogenic bacteria (PB) also. In prokaryotes, proteins of SPFH2a subfamily show highest identity to SPFH domain of Flotillin-1. Moreover, bacterial genome organization revealed that Flotillin homolog harboring SPFH2a domain exists in an operon with an upstream gene containing NFeD domain. Here, presence of RMP in PB was initially investigated in silico by analyzing the presence of SPFH2a, oligomerization domains in the concerned gene and NfeD domain in the adjacent upstream gene. After investigating 300 PB, four were found to harbor RMP. Among them, domains of Bas0525 (FlotP) of Bacillus anthracis (BA) showed highest identity with characteristic domains of RMP. Considering the global threat of BA as the bioterror agent, it was selected as a model for further in vitro characterization of rafts in PB. In silico and in vitro analysis showed significant similarity of FlotP with numerous attributes of Flotillin-1. Its punctate distribution on membrane with exclusive localization in detergent resistant membrane fraction; strongly favors presence of raft with RMP FlotP in BA. Furthermore, significant effect of Zaragozic acid (ZA), a raft associated lipid biosynthesis inhibitor, on several patho-physiological attributes of BA such as growth, morphology, membrane rigidity etc., were also observed. Specifically, a considerable decrease in membrane rigidity, strongly recommended presence of an unknown raft associated lipid molecule on membrane of BA. In addition, treatment with ZA decreased secretion of anthrax toxins and FlotP expression, suggesting potential role of raft in pathogenesis and physiology of BA. Thus, the present study not only suggest the existence and role of raft like entity in pathophysiology of BA but also its possible use for the development of novel drugs or vaccines against anthrax. [ABSTRACT FROM AUTHOR]

Ptdns(4,5)P2 is a minor structural lipid of the plasma membrane (PM), but a master regulator of PM function. Serving either as a substrate for the generation of second messengers, or more commonly as a ligand triggering protein recruitment or activation, it regulates most aspects of PM function. Understanding how this relatively simple biological macromolecule can regulate such a vast array of different functions in parallel, is the key to understanding the biology of the PM as a whole, in both health and disease. In this review, potential mechanisms are discussed that might explain how a lipid can separately regulate so many protein complexes. The focus is on the spatial distribution of the lipid molecules, their metabolism and their interactions. Open questions that still need to be resolved are highlighted, as are potential experimental approaches that might shed light on the mechanisms at play. Moreover, the broader question is raised as to whether PtdIns(4,5)P2 should be thought of as a bona fide signalling molecule or more of a simple lipid cofactor or perhaps both, depending on the context of the particular function in question. [ABSTRACT FROM AUTHOR]

Lipid modifications of mammalian proteins are widespread, modifying thousands of targets involved in all aspects of cellular physiology cellular physiology. Broadly, lipidations serve to increase protein hydrophobicity and association with cellular membranes. Often, these modifications are absolutely essential for protein stability and localization, and serve critical roles in dynamic regulation of protein function. A number of lipidated proteins are associated with diseases, including parasite infections, neurological diseases, diabetes, and cancer, suggesting that lipid modifications represent potentially attractive targets for pharmacological intervention. This review briefly describes the various types of posttranslational protein lipid modifications, proteins modified by them, and the enzymatic machinery associated with these. We then discuss several case studies demonstrating successful development of lipidation inhibitors of potential (and more rarely, realized) clinical value. Although this field remains in its infancy, we believe these examples demonstrate the potential utility of targeting protein lipidation as a viable strategy for inhibiting the function of pathogenic proteins. [ABSTRACT FROM AUTHOR]

We have analyzed the interaction of adamantyl Gb3 (adaGb3), a semi-synthetic soluble analog of Gb3, with HIV-1 surface envelope glycoprotein gp120. In this analog, which was orginally designed to inhibit verotoxin binding to its glycolipid receptor, Gb3, the fatty acid chain is replaced with a rigid globular hydrocarbon frame (adamantane). Despite its solubility, adaGb3 forms monolayers at an air-water interface. Compression isotherms of such monolayers demonstrated that the adamantane substitution resulted in a larger minimum molecular area and a more rigid, less compressible film than Gb3. Insertion of gp120 into adaGb3 monolayers was exponential whereas the gp120/Gb3 interaction curve was sigmoidal with a lag phase of 40 min. Adding cholesterol into authentic Gb3 monolayers abrogated the lag phase and increased the initial rate of interaction with gp120. This effect of cholesterol was not observed with phosphatidylcholine or sphingomyelin. In addition, verotoxin-bound adaGb3 or Gb3 plus cholesterol was recovered in fractions of comparable low density after ultracentrifugation through sucrose-density gradients in the presence of Triton X-100.The unique biological and physico-chemical properties of adaGb3 suggest that this analog may be a potent soluble mimic of Gb3, providing a novel concept for developing GSL-derived viral fusion inhibitors.

Membranes are an important signalling platform in plants. The plasma membrane is the point where information about the external environment must be converted into intracellular signals, while endomembranes are important sites of protein trafficking, organization, compartmentalization, and intracellular signalling. This requires co-ordinating the spatial distribution of proteins, their activation state, and their interacting partners. This regulation frequently occurs through post-translational modification of proteins. Proteins that associate with the cell membrane do so through transmembrane domains, protein-protein interactions, lipid binding motifs/domains or use the post-translational addition of lipid groups as prosthetic membrane anchors. S-acylation is one such lipid modification capable of anchoring proteins to the membrane. Our current knowledge of S-acylation function in plants is fairly limited compared with other post-translational modifications and S-acylation in other organisms. However, it is becoming increasingly clear that S-acylation can act as more than just a simple membrane anchor: it can also act as a regulatory mechanism in signalling pathways in plants. S-acylation is, therefore, an ideal mechanism for regulating protein function at membranes. This review discusses our current knowledge of S-acylated proteins in plants, the interaction of different lipid modifications, and the general effects of S-acylation on cellular function. [ABSTRACT FROM AUTHOR]

The accumulation of cholesterol is a general feature of cancer tissue, and recent evidence suggests that cholesterol plays critical roles in the progression of cancers, including breast, prostate, and colorectal cancers. The dysregulation of metabolic pathways, including those involved in cholesterol biosynthesis, is implicated in tumor development and cancer progression. Lipid rafts are highly dynamic cholesterol-enriched domains of the cell membrane, involved in various cellular functions, including the regulation of transmembrane signaling at the cell surface. It was recently demonstrated that lipid rafts also play critical roles in cancer cell adhesion and migration. This review focuses on our current understanding of how cholesterol regulation, lipid rafts, and dysregulated cholesterol biosynthesis contribute to cancer development and progression, and the therapeutic potential of cholesterol lowering for cancer prevention and treatment. [ABSTRACT FROM AUTHOR]

Although proteins and lipids have been assumed to be distributed homogeneously in the plasma membrane (PM), recent studies suggest that the PM is non-uniform, forming a structure that includes a number of lateral domains enriched in specific components, such as sterols, sphingolipids, and integral and surface-bound proteins. These domains are called microdomains and are considered the platform for biochemical reaction centers involved in various physiological processes. Microdomains can be extracted as detergent-resistant membrane (DRM) fractions, and such fractions have been isolated from some plant species and used for proteomic and other biochemical characterizations to gain understanding of microdomain functions. Profiling sterol-dependent proteins using a putative microdomain-disrupting agent suggests specific lipid-protein interactions in microdomains. Furthermore, in some plant species, DRM proteomes dynamically respond to biotic and abiotic stresses. Taken together, these results suggest that DRM proteomic studies provide important information for understanding microdomain physiological functions that are critical to the successful prosecution of a plant’s life cycle, particularly in its development and stress responses.

Abstract: Biological membranes organize and compartmentalize cell signaling into discrete microdomains, a process that often involves stable, cholesterol-rich platforms that facilitate protein–protein interactions. Polarized cells with distinct apical and basolateral cell processes rely on such compartmentalization to maintain proper function. In the cochlea, a variety of highly polarized sensory and non-sensory cells are responsible for the early stages of sound processing in the ear, yet little is known about the mechanisms that traffic and organize signaling complexes within these cells. We sought to determine the prevalence, localization, and protein composition of cholesterol-rich lipid microdomains in the cochlea. Lipid raft components, including the scaffolding protein caveolin and the ganglioside GM1, were found in sensory, neural, and glial cells. Mass spectrometry of detergent-resistant membrane (DRM) fractions revealed over 600 putative raft proteins associated with subcellular localization, trafficking, and metabolism. Among the DRM constituents were several proteins involved in human forms of deafness including those involved in ion homeostasis, such as the potassium channel KCNQ1, the co-transporter SLC12A2, and gap junction proteins GJA1 and GJB6. The presence of caveolin in the cochlea and the abundance of proteins in cholesterol-rich DRM suggest that lipid microdomains play a significant role in cochlear physiology. Biological significance: Although mechanisms underlying cholesterol synthesis, homeostasis, and compartmentalization in the ear are poorly understood, there are several lines of evidence indicating that cholesterol is a key modulator of cochlear function. Depletion of cholesterol in mature sensory cells alters calcium signaling, changes excitability during development, and affects the biomechanical processes in outer hair cells that are responsible for hearing acuity. More recently, we have established that the cholesterol-modulator beta-cyclodextrin is capable of inducing significant and permanent hearing loss when delivered subcutaneously at high doses. We hypothesize that proteins involved in cochlear homeostasis and otopathology are partitioned into cholesterol-rich domains. The results of a large-scale proteomic analysis point to metabolic processes, scaffolding/trafficking, and ion homeostasis as particularly associated with cholesterol microdomains. These data offer insight into the proteins and protein families that may underlie cholesterol-mediated effects in sensory cell excitability and cyclodextrin ototoxicity. [Copyright &y& Elsevier]

Sialic acid-containing glycosphingolipids, gangliosides are highly expressed in human cancer cells and regulate cell signals transduced via membrane microdomains. Generally, disialyl gangliosides enhance tumor phenotypes, while monosialyl gangliosides suppress them. In particular, gangliosides GD3 and GD2 are highly expressed in melanomas and small cell lung cancer cells, and their expression cause increased cell growth and invasion. In osteosarcomas, expression of GD3 and GD2 also enhanced cell invasion and motility, and caused increased phosphorylation of focal adhesion kinase and paxillin. In addition to focal adhesion kinase, Lyn kinase was also activated by GD3/GD2 expression, leading to the phosphorylation of paxillin. In contrast with melanoma cells, osteosarcomas showed reduced cell adhesion with increased phosphorylation of paxillin. Thus, increased expression of GD3/GD2 caused enhanced activation of signaling molecules, leading to distinct phenotypes between melanomas and osteosarcomas, i.e. increased and decreased adhesion activity. Thus, whole features of glycolipid-enriched microdomain/rafts formed in the individual cancer types seem to determine the main signaling pathway and biological outcome. [ABSTRACT FROM AUTHOR]

Carbohydrates on the glycoproteins and glycosphingolipids expressed on the cell surface membrane play crucial roles in the determination of cell fates by being involved in the fine tuning of cell signalling as reaction molecules in the front line to various extrinsic stimulants. In glycoproteins, modification of proteins is performed by substitution of sugar chains to one or multiple sites of individual proteins, leading to quantitative and qualitative changes of receptor functions in the cell membrane. As for glycosphingolipids, majority of them consist of two moieties, i.e. carbohydrates and ceramides, and are localized in the microdomains such as lipid rafts or detergent-resistant microdomains. They generate and/or modulate cell signals to determine the cell fates by interacting with various carbohydrate-recognizing proteins. Modes of glycosylation and mechanisms by which glycosylation is involved in the regulation of cell signals are now hot subjects in glycobiology. [ABSTRACT FROM AUTHOR]

The plasma membrane of eukaryotic cells participates in signal transduction and many other cellular events to maintain the physiological state of cells. In recent decades, much attention has been paid to membrane microdomains, called lipid rafts or membrane rafts, as signaling platforms in the plasma membrane. Lipid rafts are lateral lipid clusters enriched in cholesterol and sphingolipids in which particular molecules are concentrated and participate in membrane-mediated signaling events. Recent studies have shown a close relationship between lipid rafts and the age-associated decline and dysregulation of cellular signaling pathways, such as T-cell receptor signaling and cellular senescence-related signaling. Lipid rafts have also been implicated in senile diseases and in lifestyle-related diseases whose incidences increase with age. Geriatr Gerontol Int 2010; 10 (Suppl. 1): S41–S52. [ABSTRACT FROM AUTHOR]

Abstract: Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved post-translational modification in eukaryotes. In mammalian cells, approximately 150 proteins on the plasma membrane are attached to the cell surface by GPI anchors, which confer specific properties on proteins, such as association with membrane microdomains. The structures of lipid and glycan moieties on GPI anchors are remodeled during biosynthesis and after attachment to proteins. The remodeling processes are critical for transport and microdomain-association of GPI-anchored proteins. Here, we describe the structural remodeling of GPI anchors and genes required for the processes in mammals, yeast, and trypanosomes. [Copyright &y& Elsevier]

Specialized lipid microdomains in the cell plasma membrane, referred to as ’lipid rafts’, are enriched in sphingolipids and cholesterol and have drawn considerable interest as platforms for the recruitment of signaling molecules. Despite numerous biochemical and cellular studies, debate persists on the size, lifetime and even the existence of lipid rafts, emphasizing the need for reliable lipid probes to study in situ membrane lipid organization. In this review, we summarize our recent data on living cells using two specific probes of raft components: lysenin, a sphingomyelin- binding protein and the fluorescein ester of poly(ethyleneglycol)cholesteryl ether that labels cholesterol-rich domains. Sphingomyelin-rich domains that are spatially and functionally distinct from the GM1 ganglioside-rich domains were found at the plasma membrane of Jurkat T cells. In addition, the dynamics of cholesterol-rich domains could be monitored at the cell surface as well as inside the cells. [ABSTRACT FROM AUTHOR]

Abstract: The structural organization of the plasma membrane of eukaryotic cells is briefly revised taking into consideration the organization of proteins and lipids and the concept of microdomains, lipid rafts and detergent resistant membranes. The biochemical data available concerning the presence of microdomains in parasitic protozoa is reviewed and emphasis is given on the identification of special domains recognized by morphological approaches, especially with the use of the freeze–fracture technique. [Copyright &y& Elsevier]

Abstract: The EGF receptor is a transmembrane receptor tyrosine kinase that is enriched in lipid rafts. Subdomains I, II and III of the extracellular domain of the EGF receptor participate in ligand binding and dimer formation. However, the function of the cysteine-rich subdomain IV has not been elucidated. In this study, we analyzed the role of the membrane-proximal portion of subdomain IV in EGF binding and signal transduction. A double Cys→Ala mutation that breaks the most membrane-proximal disulfide bond (Cys600 to Cys612), ablated high affinity ligand binding and substantially reduced signal transduction. A similar mutation that breaks the overlapping Cys596 to Cys604 disulfide had little effect on receptor function. Mutation of residues within the Cys600 to Cys612 disulfide loop did not alter the ligand binding or signal transducing activities of the receptor. Despite the fact that the C600,612A EGF receptor was significantly impaired functionally, this receptor as well as all of the other receptors with mutations in the region of residues 596 to 612 localized normally to lipid rafts. These data suggest that the disulfide-bonded structure of the membrane-proximal portion of the EGF receptor, rather than its primary sequence, is important for EGF binding and signaling but is not involved in localizing the receptor to lipid rafts. [Copyright &y& Elsevier]

Metalloendopeptidases expressed in neural tissue are characterized in terms of their neuropeptide substrates. One such neuropeptide, bradykinin (BK), is an important inflammatory mediator that activates the type-2 BK receptor (B2R) on the terminal endings of specialized pain-sensing neurons known as nociceptors. Among several metalloendopeptidases that metabolize and inactivate BK, EP24.15 and EP24.16 are known to associate with the plasma membrane in several immortalized cell lines. Potentially, the colocalization of EP24.15/16 and B2R at plasma membrane microdomains known as lipid rafts in a physiologically relevant nociceptive system would allow for discrete, peptidase regulation of BK signaling. Western blot analysis of crude subcellular fractions and lipid raft preparations of cultured rat trigeminal ganglia demonstrate similar expression profiles between EP24.15/16 and B2R on a subcellular level. Furthermore, the treatment of primary cultures of trigeminal ganglia with inhibitors of EP24.15/16 led to the potentiation of several bradykinin-induced events that occur downstream of B2R activation. EP24.15/16 inhibition by N-[1( R, S)-carboxy-3-phenylpropyl]-Ala-AlalTyr- p-aminobenzoate (cFP) resulted in a 1000-fold increase in B2R sensitivity to BK as measured by inositol phosphate accumulation. In addition, cFP treatment resulted in a 31.1 ± 5.0% potentiation of the ability of BK to inhibit protein kinase B (Akt) activity. Taken together, these data demonstrate that EP24.15/16 modulate intracellular, peptidergic signaling cascades through B2R in a physiologically relevant nociceptive system. [ABSTRACT FROM AUTHOR]

Metalloendopeptidase EC3.4.24.15 (EP24.15) is a physiologically important neuropeptide-degrading enzyme involved in the metabolism of multiple neuropeptides. The mechanism of release of EP24.15 from neuronal cells is multimodal, being both constitutive and stimulatable. Previous studies have characterized stimulated EP24.15 secretion, yet little is understood concerning constitutive release of the peptidase. Utilizing the mouse hypothalamic neuronal GT1-7 cell line, we demonstrate that EP24.15 exists within lipid rafts in the plasma membrane, and that the enzyme is localized to the exofacial leaflet of lipid rafts. Further, we have found that biotinylated EP24.15 on the extracellular surface is released into the cell media in a fashion similar to constitutive release. In addition, classical and non-classical secretion pathway inhibitors were employed to understand the release of EP24.15 into surrounding cell media. The non-classical secretion inhibitor glyburide, a blocker of ATP-sensitive K+ channels, decreased the amount of constitutively released EP24.15 in cell media of GT1-7 cells. With these data, we conclude that EP24.15 association with lipid rafts on the extracellular surface precedes constitutive release of the peptidase into the extracellular milieu for its action on neuropeptides. [ABSTRACT FROM AUTHOR]