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Redox Signal. R edox signaling is increasingly regarded as an important cellular process in a variety of cellular activities, including cell proliferation 50 , 52 , , differentiation 72 , , , , , and apoptosis , , , , Despite extensive research, the exact mechanism by which redox enzymes are promptly activated by different stimuli still remains poorly understood, perhaps because enzymes such as NADPH oxidase, unlike G-protein-coupled enzymes, are not linked directly with any specific receptors.

Recently collected evidence suggests that membrane lipid rafts LRs and their platforms may represent an important mechanism by which redox signals are produced and transmitted in response to various agonists or stimuli , , , Many studies have shown that LRs or their platforms can participate in the signaling of cell apoptosis or dysfunction associated with oxidative stress during activation of various death receptors Major advances in LR redox signaling in specific cell types have been reported and reviewed by a series of excellent papers that have added much to the literature , , , This review will seek to further extend such LR redox signaling concept to different areas as a common signaling mechanism and thoroughly introduce the latest advances in its molecular mechanisms and the corresponding physiological and pathological relevance.

Some special emphasis will be put on the different patterns of LR redox signaling platforms, the different regulation of such redox signaling platforms, and their translational significance in health and diseases. In biological systems, electron-transfer processes play a key messenger role in redox signaling and it is primarily represented by reactive oxygen species ROS as a messenger that mediates or regulates cell—cell communication and intracellular signal transduction 28 , , Since these oxygen derivatives, whether they are radicals or nonradicals, are very reactive, they can oxidize or reduce other molecules in living cells or tissues.

Therefore, in general, redox signaling is often referred to as the signaling induced by ROS. However, these ROS are often called oxidants, since they can act as both oxidizing and reducing agents. In the literature, ROS, oxygen-derived species, and oxidants are used interchangeably to refer to the same substances active in a biological system , , Under physiological or pathological conditions, ROS can be produced as a basic signaling messenger to maintain cell or organ functions, or increasingly generated or released in response to various stimuli.

If the generation of ROS exceeds its removal by scavengers, the intracellular and extracellular levels of ROS will increase, leading to oxidative stress and a progression of various pathophysiological processes and respective diseases , If the level of ROS increases to even higher levels, its damaging effects, to DNAs, proteins, lipids, and glycols, become inevitable 28 , , These damaging effects of ROS are often tightly correlated together and share a common redox system responsible for the generation and scavenging of ROS molecules , These results demonstrated a concept of redox signaling Thereafter in both insulin and nerve growth factor were further demonstrated to stimulate H 2 O 2 production and therefore ROS and, in particular, H 2 O 2 were confirmed to have signaling actions.

However, because ROS have numerous pathological roles in various diseases and participate in bacteria killing and there is overwhelming evidence that antioxidants can prevent oxidative damage and thus protect against the adverse effects of oxidants, the pathological actions of ROS were largely focused in many studies over decades, which overshadowed the important signaling action of ROS under physiological conditions. During the last decade, the research of ROS as signaling molecules has taken a new turn.

It is now clear that in the biological systems ROS may act as autocrine, paracrine, or intracellular second messengers, involved in various signaling processes. Today it is understood that the signaling or damaging actions of ROS in or on cells are very much dependent on the level of oxidants in the cells or tissues There is agreement now that the biological responses to cellular or tissue ROS levels are very different and vary from physiological to pathological reactions. When a small amount of ROS is produced, they may mediate physiological redox signaling.

It is now widely accepted that ROS and, in particular, H 2 O 2 are involved in all types of signaling, including synaptic signaling , paracrine signaling , , autocrine signaling 43 , and intracellular signaling , as a mediator or modulator of signal transduction. So far, there are four common ROS, which are reportedly able to serve as secondary messengers. The role of NADPH oxidase in the normal regulation of cell functions has been well documented and is considered as one of the most important redox signaling pathways 82 , From the point of view of evolution, the formation of cell membranes has led to a separation of the protoplasm from the environment, enduing a cell with more independence and more capability of efficiently maintaining its integrity , Cells selectively uptake molecules through the plasma membrane, or secrete molecules into the external cellular environment, keeping an efficient homeostatic balance in substances exchanged.

Such membrane-mediated exchanges and regulatory activities facilitate the life of organisms and empower them to evolve to more advanced levels , , It is well known that the cell membranes are mainly composed of lipids, proteins, and glycols, in variable ratios, in different cell types , These membrane lipids mainly include phospholipids, sphingolipids, glycolipids, and cholesterol, and their chemical structures are shown in Figure 1. For many years, the role of these membrane lipids in the constitution of cells or various organelle membranes has been intensively studied, and several different membrane models developed to explain the structure of various biological membranes and their interaction with other components , , Composition of membrane lipids and their chemical structures.

Lipid rafts LRs may consist of dynamic assemblies of cholesterol and lipids with saturated fatty acid chains such as sphingolipids and glycosphingolipids in the exoplasmic leaflet of the membrane bilayer. In addition, phospholipids with saturated fatty acids and cholesterol in the inner leaflet. Here depicted are structures of two sphingolipids including sphingomyelin and glycosphingolipids GSL , cholesterol, and phospholipid-phosphatidylcholine. Since then, numerous studies have advanced our understanding of membrane biology.

This model emphasized the fluid characteristics of mosaic blocks in the cell membrane , Further studies, since, have demonstrated that sphingolipids and cholesterol-rich microdomains in the cell membrane have unique physical and chemical properties, which are able to form liquid ordered structures that float in the ocean of fluid glycerophospholipids.

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Such sphingolipids and cholesterol-rich microdomains have been found to play important roles in biological and physiological processes , , Until , Simons and Ikonen proposed, based on many studies of lipid patches or membrane microdomains in molecular trafficking in their own labs and others, the so-called LR model for cell membrane structures, based on the organization of sphingolipids and cholesterol mircodomains that can be selectively included or excluded 47 , , , They concluded that the function of such lipid microdomains is to serve as rafts for the transport of selected membranes or as relay stations in intracellular signaling — LRs were assumed to consist of dynamic assemblies of cholesterol and lipids with saturated acyl chains such as sphingolipids and glycosphingolipids in the exoplasmic leaflets of the membrane bilayer and phospholipids with saturated fatty acids and cholesterol in the inner leaflets Because long fatty acid of sphingolipids in the outer leaflets couples the exoplasmic and cytoplasmic leaflets by interdigitation and transmembrane proteins stabilize this coupling, LRs are very stable and detergent resistant , The sizes of individual LRs are thought to vary in different cell types from 50 to nm in diameter.

Given its small size, a raft may contain only a subset of all available raft proteins. It has been estimated that the number of proteins in each raft depends on its packing density, but it probably carries no more than 10—30 proteins This, in turn, suggests that raft clustering is important for transmembrane signaling amplification.

Notwithstanding the extensive research into them, even the existence of LRs is still not beyond doubt and some debates remain due to the lack of direct observations of such LR structures in living cells With the development of advanced technologies in microscopy and spectroscopy, such as scanning probe microscopy SPM , atomic force microscopy, single-particle tracking SPT , fluorescence correlation spectroscopy FCS , fluorescence resonance energy transfer FRET , and fluorescence photoactivation localization microscopy FPALM , more and more direct evidence gathered in living cells has shown that the nano-scale dynamic microdomains are rich in sphingolipid, cholesterol, and specific proteins Hancock suggests that rafts at the plasma membrane are present in nanoscale complexes, which are well below the optical resolution limits set by the diffraction of light.

This nanometer-size scale was supported by electron microscopic observations of immunogold-labeled raft markers More recently, by using near-field scanning optical microscopic techniques with localization accuracies of approximately 3 nm, a nanodomain of GPI-anchored proteins was observed concentrated in a region smaller than nm in fixed cells Other evidence obtained through variable waist fluorescence correlation spectroscopy indicates how GPI-anchored proteins, in the form of assemblages of less than nm in diameter, fluctuate on a subsecond time scale In addition, high spatial and temporal resolution fluorescence resonance energy transfers reveal a size estimate of approximately 10 nm in GPI-anchored receptors residing in temporally stable clusters Fluorescence photoactivation localization microscopy has shown a dynamically clustered nanoscale distribution of hemagglutinin , a transmembrane protein thought to be raft associated By analysis of the association between cholesterol and sphingolipids, in the assembly formation of membranes, using stimulated emission depletion microscopy, a study has revealed that, unlike glycerophospholipids, plasma-membrane sphingolipids display transient cholesterol-dependent confinement in areas of less than 20 nm, which is a typical LR structure All these lines of evidence obtained by using the most advanced techniques strongly support the idea that membrane molecular constituents form microdomains or LRs in the cell membrane of diverse cell types, suggesting, in turn, the presence of small, dynamic, and selective cholesterol-related microdomain heterogeneity or LRs in the plasma membranes of living cells.

It would appear that functioning LRs are not only present in cell membranes, but are responsible for molecular trafficking, transport, and signaling , Yet, many scientists who have failed to identify LRs in their work on living cells are not completely convinced that there are such things as LRs present in living cell membranes.

Two major molecular models are often utilized to describe and explain the nature and behavior of LRs. In the first model, LRs are considered relatively small structures enriched in cholesterol and sphingolipids within which associated proteins are likely to be concentrated In this sphingolipid-enriched model of LRs, the most prevalent component of the sphingolipid fraction in the cell membrane is sphingomyelin SM , which is composed of a highly hydrophobic ceramide moiety and a hydrophilic phosphorylcholine headgroup.

The tight interaction between the cholesterol-sterol-ring system and the ceramide moiety of the SM promotes a lateral association between the sphingolipids and the cholesterol, forming distinct microdomains. In these microdomains, cholesterol exerts a stabilizing role by filling the voids between the large and bulky sphingolipids. The cholesterol-SM interaction determines the transition of these microdomains into a liquid-ordered or gel-like phase that is the unique characteristic of LRs.

Other domains in cell membranes primarily exist in a more disordered fluid or liquid phase, precisely because of the absence of this cholesterol-SM interaction The second model of LRs, known as the shell hypothesis, views the generation of LRs as being based on protein—lipid or protein—protein interactions. According to this model, rafts are constructed of lipid shells, which, as small dynamic membrane assemblies, are formed by proteins preferentially associated with certain types of lipids.

Protein—protein interactions create larger functional units corresponding to LRs Other nonshell proteins associate with LRs by additional and new protein—protein interactions. In addition, an oligomerization of these proteins may create and stabilize large raft domains, forming LR platforms, making the formation and clustering of LRs dependent on both protein—lipid interactions and protein—protein interactions In many studies of the molecular models of LRs or the mechanisms forming LRs in cell membranes, two common questions have often been asked: i Why can sphingolipid- and cholesterol-enriched microdomains be separated from glycerophospholipid membrane bilayers and act as rafts floating in the membrane?

In trying to answer the first question, evidence is proffered showing that there are three main factors accounting for the formation of LRs and leading to their flotation in the cell membrane. First, compared to glycerophospholipid, the two hydrophobic SM chains are longer and more highly saturated, making them fully extended and tightly packed close to each other, which represents an important feature of LR assemblies , , The different arrangements between sphingolipids and phospholipids may be the key factor causing the phase separation in their combination , , More studies have shown that a different phase separation behavior can occur in the mixed system of cholesterol, leading to coexistence of classic mesophase and a new liquid ordered phase.

In such a new liquid ordered phase, lipid fatty acid chains are fully stretched and closely arranged into a gel like phase that exhibits a high degree of lateral mobility , , Second, unlike glycerophospholipids, SMs contain at least one hydroxyl group as shown in Figure 1 , which makes hydrogen bonds easy to form not only between SM molecules, but also between SM and cholesterol The formation of intermolecular hydrogen bonding significantly increases the intermolecular forces among these molecules, increasing the melting temperature of the lipid assembly and resulting in a transition of the assembly from a liquid disordered phase liquid phase , with lower melting temperatures into a liquid ordered phase gel phase with higher melting temperatures.

Conversion of SM into such liquid ordered phases separates it from the surrounding liquid disordered phase glycerophospholipids 39 , not unlike sphingolipid rafts floating in a sea of glycerophospholipids, a structural arrangement figuratively referred to as LRs. Finally, cholesterol can promote phase separation behavior. By filling the void space in the bulky sphingolipid molecules and forming hydrogen bonds with sphingolipids, cholesterol serves as a glue that packs the sphingolipid molecules into a more tightly organized assembly Because the sphingolipids required to combine with the cholesterol for the formation of the liquid ordered phase are much less than those without cholesterol, LRs in cells are formed with relatively much less membrane sphingolipids This is why both compounds are used as classical tool drugs in the area of LR research With respect to what types of proteins associate with LRs, there is considerable evidence that only those proteins with specific posttranslational modifications, such as the glycolphosphotidylinosital GPI -anchoring proteins, Src family tyrosine kinase, and the marker protein of LR, caveolin, can fuse in or dissociate from LRs , , Recent proteomic analysis has demonstrated that there are around authentic proteins detectable in LRs It was found that these proteins underwent several types of posttranslational modifications, thereby increasing their binding capacity to sphingolipids , These posttranslational modifications include GPI-anchoring, palmitoylation, and myristoylation.

Among these modifications, palmitoylation is attracting particular interest among investigators , Although most of these lipid modifications are irreversible, protein S-palmitoylation, also called as thioacylation or S-acylation, is able to reversibly attach, via thioester linkages, to carbon saturated fatty acids that have specific cysteine residues in their protein substrates , Such palmitoylation enhances surface hydrophobicity and the membrane affinity of protein substrates and thereby plays important roles in modulating protein trafficking 79 , , stability , sorting , etc.

It is now widely accepted that the proteins that undergo palmitoylation have a high propensity to be targeted into LRs. The concept of two types of LRs, namely, caveolar and noncaveolar rafts, in cell membranes, based on their structure and components, are well established. Caveolar rafts are formed in cell types that express caveolin proteins that bend to form scaffoldings that give shape and form caveolae. Although there have been numerous studies about caveolae functions, even before the establishment of a general LR concept 99 , , , the most well-studied function of caveolae has been its role as an important platform for the action of endothelial NOS eNOS and the synthesis of NO as a regulator of vascular dilation and constriction There is wide agreement that the binding of eNOS to the caveolin scaffolding can inhibit eNOS activity , whereas the absence of any caveolin expression can increase eNOS activity General consensus is also shared in the important role played by endothelium-specific expressions of eNOS and, in turn, the colocalization of eNOS with caveolins in ECs, in NO-mediated vasodilation and, thereby, blood pressure homeostasis The caveolinmediated formation of caveolae in ECs represents a form of LR clustering, which is present even under resting conditions.

In general, NOS in caveolae are constitutive and most activators of this enzyme do not alter the location of the NOS in caveolae. This is different from noncaveolar LRs, which largely depend on clustering or de-clustering in response to various stimuli. As shown in Figure 2 , caveolar and noncaveolar LRs mediate different signaling pathways, thereby participating in the regulation of different cell functions or cell responses to agonists or other stimuli , Demonstration of caveolar and noncaveolar lipid rafts and their function.

Caveolar and noncaveolar LRs may mediate different signaling pathways in different cells or even in the same cell in response to different agonists or stimuli. To see this illustration in color the reader is referred to the web version of this article at www. In addition to NOS regulation of caveolae, caveolae is also understood to play an important role in endocytotic or exocytotic transmembrane transport They can bud from the plasma membrane and fuse with intracellular organelles, including caveosomes , , or bud outward from the cell surface in exocytosis Caveolar endocytosis may well be a mechanism in the regulation of the lipid composition of the plasma membrane 60 , More important to redox signaling, recent studies have linked such caveolar raft-associated endocytosis with the formation of redoxosomes.

Therefore, LR- or caveolae-mediated endocytosis would be critical for the formation of redoxosomes , Such caveolae-mediated endocytotic processes have been shown to participate in the regulation of cell functions such as ion channel activities, cell polarization, molecular metabolism, recycling, and membrane repair 60 , — , , , , , According to current understanding, caveolae and noncaveolar LRs may mediate different signaling pathways, participating in the temporal-spatial regulation of the consequent cell responses even in the same type of cells.

Despite different signaling functions, the lipid components in caveolar or noncaveolar rafts are difficult to differentiate using common LR research techniques.

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Yet, there is considerable evidence that while some cell types have only caveolar or noncaveolar membrane rafts, some cell types may have both in their plasma membranes Numerous studies have been done to clarify the association of NOS with caveolae and noncaveolar rafts. They, in turn, have shed vital light on the complex features of such membrane structures as functional units. As mentioned above, the formation of caveolae may be associated with NO production and endocytosis in ECs , However, eNOS is also found in noncaveolar LRs and the formation of caveolae promotes interfacing or juxtaposing of NOS with other signaling partners such as caveolin-1, dynamin-2, calmodulin, heat shock protein 90, and akt There is evidence that although caveolin-1 is important to the formation of caveolae, this protein exerts an inhibitory action on NOS activity.

In fact, the formation of caveolae appears to play a critical role in clustering or juxtaposing various signaling components for NOS production. From this perspective, caveolinmediated formation of caveolae clearly represents a special form of LR clustering, which is constitutive and present even under resting conditions.

Noncaveolar LRs are clustered in response to agonists or stimuli. With respect to NADPH oxidase, its subunits have also been identified in caveolar and noncaveolar LRs of certain cell types studied , Like NOS, the distribution of NOX in both LRs and caveolae may also mediate different signaling pathways, participating in the temporal—spatial redox regulation of cell functions in different or even same type of cells, although in response to agonists or stimuli.

For example, in vascular smooth muscle VSM cells there is strong evidence that NADPH oxidase subunits are colocalized with caveolin-1, indicating an association of this enzyme with caveolae , Different from caveolae, LRs clustering of these noncaveolar LRs are not constitutively present, but occur only upon stimulations In spite of the difficulty in pinpointing classical LRs with SM in living cells, ceramide-enriched membrane domains are well documented. The biophysical properties of ceramide molecules predict a tight interaction of ceramide molecules with each other, resulting in the formation of stable and tightly packed ceramide-enriched membrane microdomains that spontaneously fuse to form large ceramide-enriched membrane macrodomains or platforms.

Although in a broad sense, the ceramide microdomains are also called LRs, it should be noted that ceramide-enriched membrane platforms or macrodomain can be formed without the presence of classically defined rafts, namely, the small structures enriched in cholesterol, sphingolipids, and associated proteins.

Ceramide-enriched membrane platforms are often conveniently used to describe the signaling mechanism related to these special membrane lipid platforms. Ceramide is generated in the biological membranes either by hydrolysis of SM, catalyzed, in turn, by various sphingomyelinases SMase or by a de novo ceramide synthase pathway. Both SMase and de novo synthesis—derived ceramides have been shown to be involved in cell signaling. Among SMases, acid SMase ASMase has been considered as the major enzyme responsible for the formation of ceramide-enriched membrane platforms.

The acid SMase is present locally within secretory vesicles, which are mobilized, on stimulation, to fuse with the cell membrane 81 , There is evidence that ASMase may also be found locally in lysosomal vesicles and that their activation and fusion with the cell membrane are associated with the functional integrity of lysosomes. Disturbance of lysosomal functions abolish the formation of ceramide-enriched membrane platforms associated with ASMase activation , The structure of these ceramide rafts or platforms is similar to classical SM rafts with cholesterol serving, on the one hand, as a spacer between the hydrocarbon chains of ceramide and, on the other, as dynamic glue that keeps the raft assembly together.

Cholesterol also provides partitions between the raft and the nonraft phase, having a higher affinity to raft sphingolipids ceramide here than to unsaturated phospholipids. This would appear to be confirmed by the fact that removal of raft cholesterol leads to dissociation of most proteins from the rafts, rendering them nonfunctional.

During ceramide formation, ASMase hydrolyzes SM to release choline without affecting the hydrocarbon chains that remains in the ceramide, suggesting, in turn, that cholesterol is an important component in ceramide rafts or platforms 46 , Although the constituents and the exact function of LRs inside the cell remain poorly understood, there is considerable evidence that LRs may also be present in intracellular membranes including endoplasmic reticulum membranes 21 , 47 , , , , Golgi apparatus , endosomes , , , lysosomes , , and mitochondria 63 , Studies have shown that the concentration of sphingolipids and sterols increase along the biosynthetic pathway from the endoplasmic reticulum ER to the trans-Golgi network TGN.

Such occurrence of sphingolipids and sterols may lead to functional raft clustering in these organelles, probably determining the nature of the molecular sorting, trafficking and recycling within the cells The Golgi apparatus was the first organelle demonstrated to have functional rafts that play a vital role in sorting molecules In this respect, apical sorting of GPI-anchored proteins in polarized epithelial cells has been the subject of intense research 47 , , , , which, in turn, have shown that GPI-anchored proteins associate with detergent resistant membranes DRMs during their passage through the Golgi apparatus and perturbation of this association by cholesterol or sphingolipid depletion results in impaired transport or altered polarity of the GPI-anchored proteins 47 , , , In addition, Golgi LRs have been reported to participate in the maintenance of Golgi structures and functions.

If the cholesterol balance of cells is changed, Golgi morphology and intra-Golgi protein transport may be dramatically altered , , These proteins are sorted and processed by the LRs and are then transported from the ER to the Golgi compartments 21 , 47 , , , It is assumed that the role of rafts in ER sorting has to do with its stabilizing role in the association of GPI proteins with the ER membrane.

In studies of the prion protein PrPC, also a GPI-anchored protein, it was demonstrated that perturbation of ER microdomains affects the folding of the immature protein and increases misfolding of some ER-localized mutants. Therefore, LRs on the ER may well contribute to the regulation or conformation of the PrPC and its dysfunction may be a key mechanism of neurodegenerative diseases known as Prion diseases LRs have been identified in endosomes and lysosomes , , The important roles LRs play in the endosomal recycling pathways are well known.

Raft-dependent internalization is one of the important mechanisms for the formation of endosomes, where membrane molecules and proteins are processed, transported, or metabolized. Increasing evidence has been found that LRs are present in the membrane of lysosomes. However, the mechanisms mediating the formation of LRs in lysosomal membranes and the functional relevance of such lysosomal LRs are still poorly understood. Pathologically, however, LRs are known to accumulate in late endosomes or lysosomes in patients with lysosomal storage diseases , How such pathological changes in lysosomal LRs occur remains unknown.

With respect to LRs in mitochondria, some studies have reported that mitochondria do not contain LRs and that LRs do not contain mitochondrial proteins These studies have used quantitative proteomics and multiple subcellular fractionation procedures to examine, from several angles in different cell types, whether mitochondrial proteins are in LRs. Some studies found no rafts in mitochondria and no mitochondrial proteins in cell surface rafts However, other studies have demonstrated that LR structures are detectable in mitochondria.

In such LR or LR-like domains, multiple proteins, such as GD3, the voltage-dependent anion channel-1, and the fission protein hFis, are enriched. Functionally, it is presumed that LRs in the mitochondrial complex drive mitochondrial fission, where catalytic domains are provided to associate or cleave related molecules. Disturbance of the framework of such a mitochondrial complex may impair fission and apoptosis. It has been suggested that mitochondrial LRs may represent essential activating platforms where mitochondria-mediated events determine cell survival or death 63 , It is widely accepted that the function of LRs are dependent on the formation of macrodomains or platforms, irrespective of whether they are formed or driven by SM-cholesterol and ceramide-ceramide interactions, as postulated by the sphingolipid model or alternatively by the protein—protein interactions in the shell protein model The fact that LRs, in both surface and intracellular membranes, are able to form membrane lipid platforms, begs the question whether the clustering of membrane LRs may actually produce important signaling platforms instead of being mere silent building blocks 9 , These membrane-signaling platforms play important roles in the transmembrane signaling in a variety of mammalian cells.

Here, initiation of intracellular signaling cascades is associated with aggregation or reduction of cell surface receptors through LR clustering in the plasma membrane , This completes the transmembrane signaling process 9 , 40 , , Recent studies have indicated that several death receptors, including tumor necrosis factor receptors TNFR , Fas, and death receptor DR 4 and 5, produce their apoptotic effects through this mechanism , During LR clustering, aggregated receptors or other signaling molecules are either constitutively located in the LRs or translocated by transporters or recruiters upon stimulations 45 , This dynamic clustering of lipid microdomains may represent a critical common mechanism in transmembrane signal transduction.

LRs platforms usually contain different proteins, including different signaling molecules and crosslinkers or enzymes , There is considerable evidence that LR clustering is formed as a ceramide-enriched membrane platform, where the ceramide production or enrichment is from SMase catalyzed cleavage of SM cholines in individual LRs , However, ceramide-enriched membrane platforms might also be formed without the presence of classically defined LRs simply through a fusion of several ceramide molecules. These ceramide molecules can come from LRs or other membrane fractions.

LR clustering or platform formations, especially ceramide-enriched ones are responsible for the regulation of a number of widely varied biological processes in different cells, including cell growth, differentiation and apoptosis, T-cell activation, tumor metastasis, and neutrophil and monocyte infiltration The clustering of receptor molecules within ceramide-enriched membrane platforms might well have several important functions such as the aggregation in close proximity of many receptor molecules , facilitation of the transactivation of signaling molecules associating or interacting with a receptor, and the amplification of the specific signal from activated receptors.

On the other hand, the formation of ceramide or ceramide platforms at the erythrocyte surface may partially contribute to the scrambling of the cell membrane but not assembling, leading to eryptosis after a second different stimulus such as osmotic shock. Such eryptosis may be linked to apoptotic pathways via ceramide, which, in turn, may be causally linked to local oxidative stress. This may represent another type of LR redox signaling in erythrocytes , There are many different LR signaling platforms that are formed or present in mammalian cells. These LR signaling platforms may work on different type of cells, mediating or regulating cellular activities and cell functions.

Given the stated focus of this review to be on LR redox signaling platforms, the following sections will discuss the formation and regulation of this LR signaling platform and explore related physiological and pathological relevances. Detailed information about the structural and functional nature of this family of enzymes will help understand how LR redox signaling is associated with this enzymatic system under both physiological and pathological conditions.

NADPH oxidase is a six-subunit multiprotein complex, first found abundantly expressed in phagocytic cells.

Both the structure and function of phagocytic NADPH oxidase have been thoroughly studied and are well understood. For example, it is now well known that the catalytic subunit gp91 phox also known as NOX2 and regulatory subunit p22 phox , located in the cell membrane, form heterodimers also known as flavin cytochrome b , whilst other regulatory subunits, including p47 phox , p40 phox , p67 phox , and the small G protein Rac small GTPase Rac , are located in the cytoplasm 5 , 18 , Although NOX2 is a phagocytic isoform of NOX, there is increasing evidence suggesting that NOX2 is also expressed in the nonphagocytes, including neurons, cardiac cells, skeletal muscle cells, liver cells, endothelial cells, B lymphocytes, epithelial cells, and hematopoietic cells , Under physiological circumstances, the nonphagocytic NOX expression is merely very low and its activity is maintained at a very low level.

Unlike the ROS produced in phagocytes that are mainly involved in host defense, the ROS produced in nonphagocytes primarily serve as a signaling messenger, which directly or indirectly act on the downstream intermittent or effector proteins, such as protein kinase, protein phosphatase, and various transcription factors. In this way, ROS participate in many cellular activities and cell functions, including cell proliferation and differentiation However, upon stimulation of specific agonists, such as angiotensin II Ang II , the platelet-derived growth factor PDGF , an expression of nonphagocytic NOXs, appears to be highly upregulated, although through several intracellular redox-related signaling pathways as mitogen activated protein kinases P38MAPKs , adenylate kinase AKT , and others 29 , 31 , In terms of molecular structure, NOX proteins can be divided into two major domains: i the N terminal hydrophobic transmembrane domain and ii the C terminal flavin-binding domain.

The flavin-binding domain also has some homology with a number of flavin adenine dinucleotide FAD -binding proteins, including cytochrome P reductase and ferredoxin-NADP oxidoreductase NOX family proteins have a molecular weight between 56, and 73, Da, all possessing six transmembrane domains. The NOX1 gene is located on X chromosomes and expressed mainly in the colon 5 , , VSM, the uterus, prostate, osteoblasts, and cells in the outer retina 5 , It, however, has also been detected in many other cells.

The human NOX3 gene is located on chromosome 6.

NOX3 also has a low expression level in some other tissues, such as the fetal spleen, kidney, skull, and brain NOX4, expressed primarily in adult kidneys, is possibly one of the renal oxygen-sensitive sensors 36 , In addition, NOX4 mRNA have also detected in other cells such as endothelial cells, smooth muscle cells, and fibroblasts, but only at a low expression level in monocytes.

NOX5 was found in all embryonic tissues, although with a very low expression level in the ovaries, placenta, and the pancreas However, low levels of expression were also shown recently in other tissues, such as the salivary glands, bronchus, lung, and prostate. DUOX2 were found to be mainly expressed throughout the digestive tract 11 , 92 , , Interestingly for the discussion here, almost all NOXs were demonstrated to have some structural or functional link to, or relationship with, LRs.

Indeed, many studies, in house and outside, have demonstrated that LRs even provide the driving force that promotes the assembling of NOX with other NADPH oxidase subunits or cofactors 26 , 27 , , , , , , , , , , As shown in Figure 3 , the assembly of the active NADPH oxidase phagocytic requires translocation of cytosolic subunits p47 phox and p67 phox , as well as Rac to the plasma membrane, where these subunits interact with gp91 phox and p22 phox , associating with other cofactors in the membrane to form a functional enzyme complex.

Here again, electron transfer involves cytosolic NADPH binding to gp91 phox and releasing two electrons. In the assembly and activation process of NADPH oxidase, the p47 phox translocation is a key step, and to some extent the marker for the event, since it is the first subunit translocated during the assembly process of these enzyme subunits. However, for a long time it was unknown how p47 phox translocation and subsequent assembly of other NADPH oxidase subunits occurred in the cell membrane. Even today, the driving force or physical platform upon which NADPH oxidase functions as an active enzyme complex is still unknown.

As noted above, the LR clustering or formation of LR macrodomains or platforms may represent an important mechanism mediating this assembly or activation process of NADPH oxidase. Upon stimulation, p47 phox is phosphorylated and translocated to the membrane. NADPH oxidase subunits are aggregated in the membrane to form a functional enzyme. With respect to the assembly and activation of other NOXs, there is no consensus whether they all, like phagocytic NOX, need subunits or cofactors.

Some reports have indicated that NOX1 and 4 also require all subunits and cofactors to assemble into an active enzyme complex 48 , 64 , , , , However, many other studies have reported that nonphagocytic NOXs may function without a similar assemblage as phagocytic NOX , , Figure 4 summarizes different types of NOX and their working models, where some differences among these NOXs can be seen Major Nox isoforms and their proposed model of activation. In comparison, different NOXs may work in the same way as phagocytic NOX, which need the assembly of all subunits and cofactors, or in different way as phagocytic NOX, which nonphagocytic NOXs may be functioning without assembling other subunits or cofactors.

It has been reported that NADPH oxidase exists in four different states: resting, primed, activation, and inactivation states Reference has already been made above to the NADPH oxidase activity that is regulated by its subunit phosphorylation. Most of these triggering factors act through the cell surface receptors to interact with the oxidase due to protein kinase C PKC -dependent phosphorylation of p47 phox The conformational rearrangement of p47 phox drives the cytosolic subunit to translocate to the plasma membrane In most cases, interaction between p47 phox and p22 phox promotes p67 phox and p40 phox integration with Cytob As a rapid onset triggering factor, however, PAF causes phosphorylation of p67 phox , p40 phox , and Rac 2, but not phosphorylation of p47 phox.

Phosphorylation of p67 phox is, however, necessary not only for its own translocation, but also for the translocation of p40 phox and Rac 2 to the plasma membrane After LPS incubation with neutrophils, Cyto b is translocated to the plasma membrane, and p47 phox phosphorylation and translocation are increased, respectively. In this regard, angiotensin II has been reported to induce the expression of p47 phox , p67 phox , gp91 phox , and p22 phox in skeletal muscle cells or other cells that increases the NADPH oxidase activity Numerous studies have demonstrated that various subunits or cofactors can be upregulated or downregulated by different stimuli such as cytokines, inflammatory factors, hormones, autocrines, paracrines, physical stress, and some drugs, which may be involved in transcriptional or posttranscriptional regulation of gene expression and translational or posttranslational regulation of proteins 12 , Reported SOD1 levels, for example, in LRs fractions were much higher than that in other areas of the plasma membrane.

These results support the view that in aggregation the LRs may play an important role for the SOD1 actions 6 , It is assumed that localization and subsequent aggregation of SOD1 in LRs could affect cellular functions as well as the interplay between different cell types, as LRs are rich in receptors and the signaling molecules necessary for cell—cell communications In neutrophils, proteomic analysis 90 has found catalase in LR fractions that play critical roles in redox signaling by cleavage of H 2 O 2.

Although some studies have demonstrated that LR-associated catalase may be related to peroxisome biogenesis, the function of this catalase association with LRs remains largely unknown. It is possible that LRs in hepatic peroxisomal membrane cells are able to help catalase sorting and distribution to different compartments of these cells, assigning them an important role in hepatocyte proliferation and lipid metabolism.

Given that hepatic caveolin-1 plays an important role in liver regeneration and lipid metabolism, caveolae with catalase may be critically involved in this liver regeneration and lipid metabolism. However, recent studies found that the absence of caveolin-1 did not affect the peroxisomal location of catalase in mouse liver. It seems caveolin-1 is not required for peroxisome biogenesis, whereas other types of peroxisomal LRs are required Obviously more research and thinking needs to be invested into the formation and function of LR-associated catalase complexes.

Although it is not yet extensively studied, thioredoxin has also been reported as a LR-associated protein. There is convincing evidence that LRs may mediate the actions of TRX on leukocyte—endothelial cell interaction related to redox regulation during inflammation. TRX is a ubiquitous protein with a redox-active disulfide that functions in concert with NADPH and TRX reductase to control the redox state of cysteine residues of different oxidant-targeted proteins. Given the antioxidant role of TRX, the LR—mediated role of TRX in the interaction between leukocytes and endothelial cells may importantly regulate inflammatory responses through counteracting oxidative stress and ROS In particular, a TRX mutant, TRX-C35S with replacement of cysteine 35 by serine , was found to bind rapidly to the cell surface and be internalized into the cells through LRs in the plasma membrane.

This indicates that the cysteine at the active site of TRX is important for the internalization and signal transduction of extracellular TRX through LRs , In addition to the association of LRs with ROS-producing or scavenging enzymes, another noteworthy point in LR-associated signaling molecules is the help LRs give to molecules aggregation, gating, or activation and their downstream impact on redox-sensing or enhancement of effector responses to redox signaling.

Among these molecules, a currently identified redox-sensitive protein-transient receptor protein TRP is particularly noteworthy. TRPs are a family of voltage-independent nonspecific cation-permeable channels. Perhaps these TRP channels are directly gated or influenced by the formation of LR platforms and therefore their redox-sensing function are altered. This increased channel activity may lead to enhanced redox sensitivity of the channels, exerting an important redox regulation or resulting in pathologic consequences in different cells The preceding pages have provided some insights into the role of LRs in mediating or modulating redox signaling.

On the other hand, there is increasing evidence indicating that the formation of LR—derived signaling platforms can also be altered or regulated by redox molecules. In addition, various ROS species were found to influence LR signaling or function through their actions on many LR constituents such as ceramide production, cholesterol, and related raft proteins 81 , ASMase, which play a key role in the formation of ceramide-enriched membrane platforms have been extensively studied. ROS generation, for example, is known to be intimately involved in the activation of the enzyme in response to various stimuli.

Pretreatment of neutrophils with the antioxidants N-acetylcysteine NAC and desferrioxamine significantly inhibited the downstream ASMase activities, such as ceramide generation and CD95 clustering. A new model proposed by Gulbins et al. Based on this model, the free C-terminal cysteine of ASMase can be modified and lost by the actions of ROS, wherein a zinc coordination in this enzyme is altered, leading to the activation or inhibition of the enzyme. Confirmation of the links between redox regulation of ceramide-enriched membrane platforms and glioma chemotherapy illustrated this.

By transfection of human or murine glioma cells with ASMase, marked sensitization of the glioma cells to gemcitabine and doxorubicin occurred, accompanied by increased activation of ASMase, elevated ceramide levels and enhanced formation of ceramide-enriched membrane platforms.

Native low density lipoprotein promotes lipid raft formation in macrophages

Taken together, ROS also regulates the formation of LR signaling platforms and therefore LRs and ROS may constitute an amplification of signals in different biological membranes, insuring the efficiency of signal transduction. Such feedforwarding regulation will be further discussed below in the regulation of LR redox signaling platforms. The most important factors in the detection of LR redox signaling platforms are the colocalization of lipid components and aggregated or recruited NADPH oxidase subunits or other molecules.

Individual LRs on the cell membrane are too small suggested to be around 50 nm in diameter to be resolved by standard light microscopy, but once several separate small LRs were clustered upon stimulation, these LR clusters could be observed as patches or spots under microscope Therefore, fluorescent staining and confocal microscopic imaging of LR patches or spots on the cell membrane is the most frequently used method to identify the formation of LR signaling platforms including LR redox signaling platforms.

The fluorescence labeling of the B subunit of cholera toxin CTXB is widely used as a common LR marker to perform colocalization with some LR-associated redox molecules such as NOXs and other subunits including p47 phox , p21 phox , p67 phox and others. In addition, given that ceramide-enriched signaling platforms are considered as another type of LRs, anticeramide antibodies can also be used as a marker of LRs or sphingolipids to detect LR-associated redox enzymes or related molecules Fluorescence resonance energy transfer FRET is a phenomenon that occurs between a fluorophore pair, donor and acceptor e.

The fluorophore pair both share the same characteristics in the transfer of energy from the donor to the acceptor, namely the overlap of the emission wavelength of the donor with the excitation of the acceptor's wavelength The two key factors determining the occurrence of FRET are molecular orientation and distance between the molecules.

It is proposed that FRET can only take place between two molecules within 7—10 nm range. Detected FRET generally indicates that two molecules are closely located, allowing them to generate an energy transfer from one to the other that leads to molecular reactions. FRET analysis, with resolutions believed to be at lower than 10 nm of separations between the two molecules, may significantly enhance [colocalization using regular confocal microcopy requires a separation of greater than nm ] the resolution of common confocal microscopic observations.

Both donor and acceptor bleaching protocols can be employed to measure the FRET efficiency. As described elsewhere , , , , acceptor bleaching protocols first prepared prebleaching acceptor images followed by increases of the excitation wavelength of the acceptor TRITC laser intensity from 50 to 98 for 2 min bleaching the acceptor fluorescence.

After the intensity of the excitation laser of the acceptor was adjusted back to 50, the postbleaching image was then taken. The FRET image was obtained by subtracting the prebleaching image from the postbleaching image in blue. Some examples of such confocal microscopic colocalization and FRET detections in endothelial cells are presented in Figure 5.

Panel A shows colocalization of CTXB and gp91 phox as indicated by yellow spots or dots in overlaid images. Confocal microscopic colocalization and FRET detection. Biochemically, the method most often used for detection of LRs is the flotation of DRMs in combination with Western blots to identify associated proteins or receptors in LR fractions During sucrose gradient centrifugation, DRMs complexes or detergent insoluble glycolipid-enriched domains DIG can float to low-density fractions and reinforce the integrity of LRs structure.

These LR fractions contain abundant raft proteins and therefore analyzing the raft proteins in DRMs by immunoblotting provides a reliable and simple means for identifying possible LR components, especially LR-associated proteins such NOXs or related subunits or cofactors Further, if large scale proteomic analyses could reach sufficient resolutions and sensitivities, in combination with proteomic techniques developed recently, this membrane flotation technique could help identify many as yet unobserved molecules including receptors, enzymes, regulators and adaptors Recently, there have been some challenges to the use of DRMs 4 , and their possible artifacts, such as LR fractions.

The procedure for the isolation of nondetergent MR fractions has been developed and used , significantly increasing the sensitivity and specificity of isolated LR proteins or components. In addition, using 3-layer gradient centrifugation for isolation of LR fractions, researchers have succeeded in separating noncaveolar and caveolar fractions in classical DRMs flotations , A modified nondetergent 4-layer gradient centrifugation is now used to isolate LR fractions.

This method separates, respectively, light low density fractions, heavy low density fractions and other high density fractions, which represent noncaveolar, caveolar and other fractions of membrane proteins, making it now possible to identify and separate signaling molecules or enzymes in LR clusters in both caveolar and noncaveolar compartments. Such membrane flotation will provide more and increasingly accurate information about the location of LR redox signaling platforms by detecting their distribution in different fractions.

A typical gel document using nondetergent and modified 4-layer gradient flotation and then Western blot analysis of gp91 phox is presented in Figure 6. Among 24 fractions, 3—6 and 10—14 represent light and heavy, low-density fractions, respectively, which correspond to noncaveolar and caveolar LRs. Under controlled conditions, interestingly, gp91 phox is present in caveolar fractions, but not in noncaveolar fractions. When the cells were treated with Fas ligand, the fractions were shifted to noncaveolar fractions. The tumor-suppressing role of Cav1 is dependent on the expression of various other molecular effectors making it a conditional function.

Negative regulation of T-cell activation and autoimmunity by Mgat5 N -glycosylation. Nature , — Regulation of cytokine receptors by Golgi N -glycan processing and endocytosis. Science , — Conversely, Cav1 has also been found to exert tumor-promoting effects that are associated with phosphorylation of its Y14 by the tyrosine kinase family members Src, cAbl and Fyn, or at S80 [61] Williams TM, Lisanti MP. There is accumulating evidence that Cav1 plays conflicting paradoxical roles in cancer development and progression [44] Patra SK.

Caveolin-1 tumor-promoting role in human melanoma. There is an obvious paradox of roles played by Cav1 in transformation of cells and their progression to the metastatic phenotype. Inactivation of Cav1 appears to be necessary for cell transformation and tumor induction, whereas its re-expression facilitates tumor progression and metastasis [44] Patra SK. Tyrosine protein kinases belonging to the Src-family Lyn, Fyn, proto-oncogene tyrosine kinase Src and c-src are common raft components, and beyond their normal role in endocytotic processes, they interact via their SH2 or SH3 domains with proteins involved in cellular adhesion and growth control.

In this way, MUC1 functions as a transforming protein by coactivating transcription of Wnt target genes. While MUC1 is an epithelial antigen and its overexpression is a feature of various carcinomas, subpopulations of red and white blood cells erythroblasts and lymphoblasts are also MUC1 positive. Although beyond the scope of this article, an outline of available techniques and their pros and cons should be given at this point see also Table 2.

Generally, two principle strategies in the profiling of complex proteomes are applicable: the historically older top-down or gel-based approaches, where reduction of sample complexity is achieved by 2-DE separation at the protein level prior to in-gel digestion and liquid chromatography-mass spectrometry LC-MS ; and the bottom-up or shotgun approaches, where initial in-solution digestion is followed by 2D-LC at the peptide level prior to LC-MS. Both approaches have their merits, however, the shotgun technique is clearly favorable with respect to membrane proteomics in general, and in particular with respect to the profiling of integral lipid raft proteins.

It has been demonstrated by numerous studies that the shotgun approach results in much higher numbers of identified proteins. To understand this finding, it should be considered that in 2-DE settings, integral membrane proteins are largely lost, since their physicochemical characteristics hydrophobic domains, multiply-charged post-translational modifications and molecular size are not compatible with the narrow window of isoelectric focusing and polyacrylamide gel electrophoresis parameters in 2-DE.

Lipid rafts exhibit a heterogeneous population of both constitutive and fluctuating protein components. In order to characterize the components of rafts that contain any one specific protein, affinity-based enrichment strategies must be employed. We have developed such a strategy for the protein-specific isolation of lipid raft subpopulations from plasma membranes and exosomal membranes that are characterized by the content of a specific target protein [5] Staubach S, Razawi H, Hanisch FG.

The principle of the strategy see schematic flow chart in Figure 3 is based on the use of a recombinantly expressed, epitope-tagged fusion protein that serves as a bait protein for affinity enrichment of specific raft subpopulations. Importantly, this type 1 transmembrane mini-mucin exhibits an oligo-histidine and a myc epitope-tag adjacent to the N-terminal signal peptide.

The construct was transfected into MCF-7 breast cancer cells and the exosomes were isolated from culture supernatants of cells grown in the presence of fetal calf serum that had been depleted from bovine exosomes by ultracentrifugation. We followed standard protocols for cellular exosome isolation based on three differential centrifugation steps, followed by exosome sedimentation and washing in two consecutive ultracentrifugation steps. Finally, the lipid raft fractions were subfractionated using paramagnetic beads conjugated to anti-myc antibodies to enrich the raft subpopulation containing MUC1-M2.

Recovered raft-associated proteins were precipitated with chloroform-methanol and subjected to 2-DE. The detergent-resistant membrane subfractions can be further separated from non-raft membrane fractions by sucrose-density gradient centrifugation. Cholesterol-rich rafts exhibit low buoyant density compared with non-raft membrane fractions.

In gel-based or top-down applications, the gels are stained either with a fluorescent dye, coomassie blue or an MS-compatible silver stain. Proteins identified by MS should be validated, at least for a selected panel, by Western blot analysis. However, it should be kept in mind that even validated proteins may represent contaminants, since mitochondrial or cytosolic proteins have been found in numerous studies to stick nonspecifically to affinity matrices.

Prior to raft isolation by detergent extraction in the cold, treated and untreated control cells were mixed and raft enrichment was performed by density gradient centrifugation. Each peptidic fragment from the cellular proteome was represented by two signals in the mass spectra, corresponding to the light and heavy isotopic species, discriminated on the basis of their slightly differing relative masses.

There are numerous reports in the current literature referring to cancer markers identified by quantitative or differential proteomics and these numbers increase steadily from year to year. The studies differ with respect to their focus on particular tumor types or locations and to their aims identification of tissue or serum markers and with respect to the applied technologies 2-DE, 2D-DIGE and so on.

Owing to space considerations, only a selection of representative examples can be cited in this article, highlighting specific aspects with relevance to the topic. Incidentally, only a small number of proteomic studies refer explicitly to lipid rafts in cancer. In vitro culture model systems can be valuable tools for proteomics-based identification of candidate biomarkers, if combined with suitable validation or prioritization tests.

A recent study focused on the identification of extracellular markers in the progression of breast cancer using an in vitro cell culture model and the 2D-DIGE technology. By combining experimental and in silico approaches, the authors were able to discover and prioritize candidate biomarkers that are more likely to be found in serum [77] Lau TY, Power KA, Dijon S et al. Prioritization of candidate protein biomarkers from an in vitro model system of breast tumor progression toward clinical verification.

Proteome Res. Quantitative organelle proteomics of MCF-7 breast cancer cells reveals multiple subcellular locations for proteins in cellular functional processes. A targeted proteomic approach was used to identify potential N-linked glycoprotein markers from breast cancer cell membrane fractions and to map the sites of modification [79] Whelan SA, Lu M, He J et al.

Of the total 25 N-linked glycoproteins identified in different cell lines, only three were found in all cellular models galectinbinding protein, lysosome-associated membrane glycoprotein 1 and oxygen-regulated protein. Other studies focusing on the progression aspect of cancer have used a combination of differential 2-DE proteomics and Western blot validation of the candidate biomarkers. A syngeneic cellular model for the progression of colorectal adenoma to carcinoma was analyzed in this way and revealed a panel of partially lipid raft-associated proteins villin-1, annexin A1 and Grp78 showing regulated expression [80] Roth U, Razawi H, Hommer J et al.

Differential expression proteomics of human colorectal cancer based on a syngeneic cellular model for the progression of adenoma to carcinoma. Proteomics 10 2 , — In the same context and using similar techniques, another study was based on tumor and adjacent nontumor tissue samples from colorectal cancer patients and revealed heterogenous nuclear ribonucleoprotein A1 as a potential biomarker also suitable for serotesting [81] Ma YL, Peng JY, Zhang P et al.

Heterogeneous nuclear ribonucleoprotein A1 is identified as a potential biomarker for colorectal cancer based on differential proteomics technology. Quantitative proteomic profiling of membrane fractions from human normal and cancerous colorectal tissues was performed by using the isobaric tagging with relative and absolute quantitation iTRAQ approach [82] Chen JS, Chen KT, Fan CW et al. Comparison of membrane fraction proteomic profiles of normal and cancerous human colorectal tissues with gel-assisted digestion and iTRAQ labeling mass spectrometry.

FEBS J. This shotgun approach revealed 34 upregulated and eight downregulated proteins with expression changes greater than twofold out of over total proteins identified in membranous fractions from cancer patients. While differential proteomic studies with focus on raft-associated proteins in human cancer tissue are still rare, a number of publications refer to protein compositions of cancer rafts or plasma membrane-derived vesicles, called exosomes.

Exosomes represent nanovesicles, which originate from early endosomes via a second invagination of the endosomal membrane to form multivesicular bodies MVBs. After fusion with the plasma membrane, these MVBs release their contents into the extracellular space. Exosomes have been postulated to fulfil a variety of functions, including cell communication and protein export.

Proteomics analysis of A33 immunoaffinity-purified exosomes released from the human colon tumor cell line LIM reveals a tissue-specific protein signature. Cell Proteomics 9 2 , — Of nearly identified exosomal proteins, a subset was common to human exosomes from other sources, such as urine endosomal sorting complex required for transport, tetraspanins, signaling, trafficking and cytoskeletal proteins. However, a number of proteins were host cell specific A33, cadherin, carcinoembryonic antigen, epithelial cell surface antigen [EpCAM], proliferating cell nuclear antigen, EGFR, MUC13, misshapen-like kinase 1, keratin, mitogen-activated protein kinase 4, claudins 1, 3 and 7, centrosomal protein 55 kDa, and ephrin-B1 and -B2.

Exosomes may play an important role in diagnosis and in biomarker studies in general, due to their ready isolation from human body fluids and the fact that they offer insight into protein compositions of plasma membrane subfractions derived from the respective host cells [84] Simpson RJ, Lim JW, Moritz RL, Mathivanan S. Exosomes: proteomic insights and diagnostic potential. Expert Rev. Proteomics 6 3 , — Exosomes were present in pleural fluid from patients suffering from mesothelioma, lung cancer, breast cancer and ovarian cancer [85] Bard MP, Hegmans JP, Hemmes A et al.

Proteomic analysis of exosomes isolated from human malignant pleural effusions. Cell Mol. Referring more specifically to the topic of this article, the dysregulation of lipid raft proteins and the subsequent effects on signaling and tumor progression needs to be addressed. Because upregulation of uPA and uPAR in cancer appears to potentiate the malignant phenotype, the understanding of how uPAR changes the downstream cellular proteome is fundamental.

Saldanha et al. Differential proteome expression associated with urokinase plasminogen activator receptor uPAR suppression in malignant epithelial cancer. In addition, Ahmed et al. Proteomic profiling of proteins associated with urokinase plasminogen activator receptor in a colon cancer cell line using an antisense approach. Proteomics 3 3 , — These authors demonstrated the loss of approximately proteins and quantitative differences in the expression of other proteins.

It is found in the majority of primary and metastatic transitional cell carcinomas as well as in breast cancer tissues, but not in adjacent normal tissues. Metastasis-associated C4. These results indicate that the cleavage of C4. Rescue of paclitaxel sensitivity by repression of prohibitin1 in drug-resistant cancer cells. USA 6 , — This important finding has relevance for various cancers including breast, lung and ovary cancer, since PHB1 may serve as a potential target for therapeutic strategies for the treatment of drug-resistant tumors.

Lipid rafts and clusters of apoptotic signaling molecule-enriched rafts in cancer therapy. Future Oncol. The subsequent signaling cascade results in caspase activation, formation of an apoptosome that is controlled by different pro- and anti-apoptic mitochondrial proteins, and subsequently the release of cytochrome C and the final degradation of cellular protein and DNA.

Dimerization and oligomerization of receptors for extrinsic signal transmission through the membrane were induced by binding of the respective ligands. In cancer, these receptors are often mutated or, due to an upregulated threshold, are nearly inactive. Accumulation of these receptors in proximity to lipid rafts also leads to the activation of the signaling cascade. Proteins specifically expressed in caveolar rafts of endothelial cells in neovascularized tumors could be targets for anticancer drugs.

Such targets were identified using quantitative proteomic analysis of endothelial membrane fractions from blood vessels of different organs and tumors [91] Oh P, Li Y, Yu J et al. Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue-specific therapy. Among the candidate marker proteins were aminopeptidase P and annexin Application of antibody-based fluorescently labeled nanoparticles generated against aminopeptidase P and in vivo fluorescence microscopy revealed rapid uptake of the particles by the lung epithelium via caveolae.

Cav1 downregulation inhibited caveolar uptake and proved that this transport route could be used for selective import of drugs into tumor cells. With the development of sophisticated strategies in proteome research, the chance of identifying novel biomarkers has been greatly increased.

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This is particularly true in the cancer research field, where classical immunological approaches are now complemented by qualitative and quantitative proteomics applications. In particular, the latter may become increasingly important, since they offer the possibility of differential analyses of normal versus diseased cellular states and hence the identification of regulated protein species. Proteomics as a technical platform for biomarker identification is a promising tool, in particular with respect to the analysis of cellular subproteomes, because it allows a focused view on a less complex proteome and, by fading out abundant cellular proteins, a more sensitive and deeper insight into the proteome.

Lipid rafts are an example of such a subproteome, representing highly dynamic sorting and signaling platforms of plasma membranes. These rafts should be ideal targets for identifying protein markers that fluctuate in disease cell states, such as cancer. Moreover, rafts and their distinct physical properties should also offer the possibility of targeted therapeutic applications against cancer. There is no doubt that the concept is worthy of consideration by researchers and by the pharmaceutical industry with respect to targeted cancer treatment.

Mass spectrometry-based proteomics is still a rapidly evolving field, where technological advancements can be expected to facilitate and speed up established strategies as novel techniques become applicable. The quantification of candidate biomarkers in human cancer, preferentially performed by immunoassays, will slowly be replaced by quantitative targeted proteomics. Furthermore, advancements in the laser technology within MALDI MS could enhance MALDI imaging as an attractive alternative to antibody-based histology and subsequently a technology applicable at even the single cell level, as it allows multiple biomarker imaging in parallel without the need for specific antibodies.

Key issues. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. Describes an affinity-based strategy for isolation of raft subpopulations. Skip to Main Content. Search in: This Journal Anywhere.

Advanced search. Submit an article Journal homepage. Pages Published online: 09 Jan Lipid rafts: signaling and sorting platforms of cells and their roles in cancer. Lipid rafts: signaling and sorting platforms of cells and their roles in cancer All authors. Published online: 09 January Figure 1. Alternative mucin 1 endocytosis pathways. Display full size. Figure 2. The scaffolding of signaling proteins by caveolin Figure 3.

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Steps in the affinity-based enrichment of protein-specific lipid rafts from plasma membranes or exosomes. Lipid raft formation requires scaffolding proteins The fluid mosaic model of plasma membranes has been revised in recent years to incorporate the concept that membranes exhibit detergentresistant subdomains or lipid rafts with characteristic protein and lipid compositions [1] Lingwood D, Simons K. The cancer mucin MUC1 recycles via alternative raft-associated pathways The surface expression of some apically-expressed glycoproteins in polarized epithelial cells is only transient as they either traffic back to the glycosylation machinery of the trans-Golgi network TGN for further glycosylation cycles or they are exported via exosomes Figure 1.

The roles of chaperones Heat-shock proteins Hsps demonstrate not only a cytosolic localization, but also a strong association with cellular membranes, in particular at the plasma membrane. The paradoxical role of Cav1 Caveolin-1 is an integral membrane protein found in caveolae and is essential as a scaffolding protein for the formation of these omega-shaped invaginations of the plasma membrane see earlier. Enrichment of lipid raft subpopulations by affinity chromatography-based techniques Lipid rafts exhibit a heterogeneous population of both constitutive and fluctuating protein components.

Identification of cancer markers by differential proteomics There are numerous reports in the current literature referring to cancer markers identified by quantitative or differential proteomics and these numbers increase steadily from year to year. Breast cancer In vitro culture model systems can be valuable tools for proteomics-based identification of candidate biomarkers, if combined with suitable validation or prioritization tests.

Colon cancer Other studies focusing on the progression aspect of cancer have used a combination of differential 2-DE proteomics and Western blot validation of the candidate biomarkers. Exosomes as a biomarker source While differential proteomic studies with focus on raft-associated proteins in human cancer tissue are still rare, a number of publications refer to protein compositions of cancer rafts or plasma membrane-derived vesicles, called exosomes.

Lipid raft proteins in tumor progression Referring more specifically to the topic of this article, the dysregulation of lipid raft proteins and the subsequent effects on signaling and tumor progression needs to be addressed. Expert commentary With the development of sophisticated strategies in proteome research, the chance of identifying novel biomarkers has been greatly increased. Five-year view Mass spectrometry-based proteomics is still a rapidly evolving field, where technological advancements can be expected to facilitate and speed up established strategies as novel techniques become applicable.

Table 1. Proteins identified in cancer lipid rafts. CSV Display Table. Table 2. Technical approaches to the proteome of lipid rafts. Article Metrics Views. Article metrics information Disclaimer for citing articles. People also read Article. Alex J. Laude et al. Molecular Membrane Biology Volume 21, - Issue 3. Published online: 9 Jul Maulucci et al. Free Radical Research Volume 50, - Issue sup1.

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Published online: 25 Oct Mika Hilvo et al. Clinical Lipidology Volume 7, - Issue 2. Published online: 18 Jan Stella M. Lu et al. Published online: 18 Feb Joanna M. Cordy et al. Molecular Membrane Biology Volume 23, - Issue 1. Ahmed Mohamed et al. Expert Review of Proteomics Volume 15, - Issue Published online: 13 Nov Proteomic analysis of membrane rafts of melanoma cells identifies protein patterns characteristic of the tumor progression stage.

Proteomics 8 22 , — Protein profiling of plasma membranes defines aberrant signaling pathways in mantle cell lymphoma. Proteomics 8 7 , — Subcellular proteomics of cell differentiation: quantitative analysis of the plasma membrane proteome of Caco-2 cells. Proteomics 7 13 , — Membrane microdomains and proteomics: lessons from tetraspanin microdomains and comparison with lipid rafts.

Proteomics 6 24 , — Patra SK.

1. Introduction

Impact of glycosylation and detergent-resistant membranes on the function of intestinal sucrase-isomaltase. In-solution protein digestion followed by shotgun analysis for global protein profiling. The application of mass spectrometry to membrane proteomics. Membrane proteins ride shotgun. Proteomic analysis of detergent-resistant membrane rafts. Electrophoresis 25 9 , — Comparative data on bottom-up vs top-down approaches to membrane proteomics. Proteomic analysis of plasma membrane lipid rafts of HL cells.

Proteomics 7 14 , — Qualitative profiling of melanoma cell lines derived from tumors at different stages of progression. Proteomics 4 10 , — Trypsin digestion of raft proteins presence of mitochondrial raft proteins. Lipid raft proteome reveals ATP synthase complex in the cell surface.

Proteomics 4 11 , —