Membrane lipid therapy: Modulation of the cell membrane composition and structure as a molecular base for drug discovery and new disease treatment

Nowadays we understand cell membranes not as a simple double lipid layer but as a collection of complex and dynamic protein–lipid structures and microdomains that serve as functional platforms for interacting signaling lipids and proteins. Membrane lipids and lipid structures participate directly as messengers or regulators of signal transduction. In addition, protein–lipid interactions participate in the localization of signaling protein partners to specific membrane microdomains. Thus, lipid alterations change cell signaling that are associated with a variety of diseases including cancer, obesity, neurodegenerative disorders, cardiovascular pathologies, etc. This article reviews the newly emerging field of membrane lipid therapy which involves the pharmacological regulation of membrane lipid composition and structure for the treatment of diseases. Membrane lipid therapy proposes the use of new molecules specifically designed to modify membrane lipid structures and microdomains as pharmaceutical disease-modifying agents by reversing the malfunction or altering the expression of disease-specific protein or lipid signal cascades. Here, we provide an in-depth analysis of this emerging field, especially its molecular bases and its relevance to the development of innovative therapeutic approaches.     

Proteins at membrane surfaces-a review of approaches

Membrane proteins are critical for normal cellular differentiation and function, and alterations in these proteins often leads to cell dysfunction and disease. Membrane proteomics aims to identify the membrane protein constituents, their posttranslational modifications, protein-protein interactions, and dynamics. Efforts to identify membrane proteins and elucidate their dynamics have been plagued by the challenges presented by studying water insoluble proteins that are distributed among a range of membranes in a cell and often occur at a relatively low abundance. This brief review presents a summary of the literature related to membrane proteomics with an emphasis on efforts to develop effective protocols for the enrichment of membrane proteins, particularly those located in the plasma membrane.     

Quantitative analysis of caveolin-rich lipid raft proteins from primary and metastatic colorectal cancer clones

Caveolin-rich lipid rafts (CLRs) are thickened sections of the cell membrane that are composed of the integral membrane proteins caveolins together with saturated long chain fatty acids, cholesterol and lipids. Membrane proteins - lipid raft proteins in particular - may play important roles in cell signaling and cell-cell interaction. Due to their unique structure, CLRs seem to be the preferred docking site for specific proteins involved in focal adhesion and cancer metastasis. Our objective was thus to identify and quantify CLR proteins from primary and metastatic colorectal cancer (CRC) clones. We found differential expression of nine CLR proteins from primary and metastatic CRC clones. Among the identified proteins, an immune system inhibiting protein was significantly overexpressed in the metastatic clone, while cell adhesion and transport molecules were among the overexpressed proteins in the primary clone. All the identified CRL proteins are involved in tumorigenesis, specifically metastasis, and may thus serve as therapeutic targets. A novel concept for identification and quantification of CLR proteins with label-free mass spectrometry method was specifically examined in this study. Validation of the method against immunoblotting and FACS analysis indicates that it can be applied for the identification of novel biomarkers for cancer and metastasis.     

Membrane protein analysis of human breast cancer cell line MCF-7 by different membrane washing methods

Membrane and membrane-associated proteins are rich in known or potential pharmaceutical drug targets for carcinogenesis. In order to systemically analyze membrane proteins of human breast cancer, we isolated membrane from MCF-7 cells by sequential extraction by washing with three different buffers, namely, phosphate buffer (5 mM, pH 8.0), Tris (40 mM, pH 9.5), and sodium carbonate (100 mM pH 11). The extracted proteins were separated by two-dimensional gel electrophoresis (2-DE) using cup-loading and were then analyzed by peptide mass fingerprinting (PMF). A total of 137 spots from the gels of the three procedures were successfully identified. They corresponded to 79 distinct proteins. Among them, 22 exclusive proteins belonging to each washing procedure were also found, including P-glycoprotein, endoplasmin, Stress-70 protein, ADAM 10, protein disulfide isomerase, and glutamate receptor. These results indicate phosphate buffer to be the most beneficial for enrichment of peripheral membrane proteins, and sodium carbonate is beneficial for the presentation of integral membrane proteins but usually with poor resolution. The reference maps and identified proteins will serve as a basis for the further investigation of breast cancer, especially the proteomic comparison among different cell types of breast cancer, or among the different stages in the drug interfering process of the MCF-7 cell line.     

Computational design of membrane proteins

Membrane proteins are involved in a wide variety of cellular processes, and are typically part of the first interaction a cell has with extracellular molecules. As a result, these proteins comprise a majority of known drug targets. Membrane proteins are among the most difficult proteins to obtain and characterize, and a structure-based understanding of their properties can be difficult to elucidate. Notwithstanding, the design of membrane proteins can provide stringent tests of our understanding of these crucial biological systems, as well as introduce novel or targeted functionalities. Computational design methods have been particularly helpful in addressing these issues, and this review discusses recent studies that tailor membrane proteins to display specific structures or functions and examines how redesigned membrane proteins are being used to facilitate structural and functional studies.     

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets^1-3. Since they function in complexes⁴, methods to identify and characterize their interactions are necessary⁵. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE⁶. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.     

Membranes: a meeting point for lipids, proteins and therapies

Membranes constitute a meeting point for lipids and proteins. Not only do they define the entity of cells and cytosolic organelles but they also display a wide variety of important functions previously ascribed to the activity of proteins alone. Indeed, lipids have commonly been considered a mere support for the transient or permanent association of membrane proteins, while acting as a selective cell/organelle barrier. However, mounting evidence demonstrates that lipids themselves regulate the location and activity of many membrane proteins, as well as defining membrane microdomains that serve as spatio-temporal platforms for interacting signalling proteins. Membrane lipids are crucial in the fission and fusion of lipid bilayers and they also act as sensors to control environmental or physiological conditions. Lipids and lipid structures participate directly as messengers or regulators of signal transduction. Moreover, their alteration has been associated with the development of numerous diseases. Proteins can interact with membranes through lipid co-/post-translational modifications, and electrostatic and hydrophobic interactions, van der Waals forces and hydrogen bonding are all involved in the associations among membrane proteins and lipids. The present study reviews these interactions from the molecular and biomedical point of view, and the effects of their modulation on the physiological activity of cells, the aetiology of human diseases and the design of clinical drugs. In fact, the influence of lipids on protein function is reflected in the possibility to use these molecular species as targets for therapies against cancer, obesity, neurodegenerative disorders, cardiovascular pathologies and other diseases, using a new approach called membrane-lipid therapy.     

Two-photon polarization microscopy reveals protein structure and function

Membrane proteins are a large, diverse group of proteins, serving a multitude of cellular functions. They are difficult to study because of their requirement of a lipid membrane for function. Here we show that two-photon polarization microscopy can take advantage of the cell membrane requirement to yield insights into membrane protein structure and function, in living cells and organisms. The technique allows sensitive imaging of G-protein activation, changes in intracellular calcium concentration and other processes, and is not limited to membrane proteins. Conveniently, many suitable probes for two-photon polarization microscopy already exist.     

The cytoskeletal proteins of erythrocytes

The erythrocyte membrane skeleton is composed of the number of proteins isolated and characterized. One of the major proteins of cytoskeleton is actin presented in erythrocytes in the form of short protofilaments. This review will focus on the manner of attachment of actin protofilaments to the red cell membrane, and on the relationships between skeleton membrane proteins. Membrane skeleton proteins in erythrocytes are not unique. Recently a lot of proteins similar to the red cell membrane skeleton proteins were found in a wide variety of non-erythroid cells. This fact gives the opportunity to suppose the existence of a unique protein system in erythroid and non-erythroid cells which provides the attachment of actin filaments to cell membranes and which might be the centre for the assembling of actin structures in the cortical cytoplasm.     

Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy

Membrane proteins play many critical roles in cells, mediating flow of material and information across cell membranes. They have evolved to perform these functions in the environment of a cell membrane, whose physicochemical properties are often different from those of common cell membrane mimetics used for structure determination. As a result, membrane proteins are difficult to study by traditional methods of structural biology, and they are significantly underrepresented in the protein structure databank. Solid-state Nuclear Magnetic Resonance (SSNMR) has long been considered as an attractive alternative because it allows for studies of membrane proteins in both native-like membranes composed of synthetic lipids and in cell membranes. Over the past decade, SSNMR has been rapidly developing into a major structural method, and a growing number of membrane protein structures obtained by this technique highlights its potential. Here we discuss membrane protein sample requirements, review recent progress in SSNMR methodologies, and describe recent advances in characterizing membrane proteins in the environment of a cellular membrane.     

Apoptosis-Independent Alterations in Membrane Dynamics Induced by Curcumin

Curcumin is a well-known natural compound with antiinflammatory properties. Its antiproliferative effect and ability to modulate apoptotic response are considered essential in cancer therapy. The physicochemical properties of curcumin suggest membranous localization, which prompted an investigation of the mechanisms of membrane disturbances evoked by curcumin. We chose the erythrocyte as a convenient model for studying membrane effects of curcumin and showed its nonspecific, apoptosis-independent way of action. Curcumin was found to expand the cell membrane, inducing echinocytosis. Changes in cell shape were accompanied by transient exposure of phosphatidylserine. Membrane asymmetry was recovered by the action of aminophospholipid translocase, which remained active in the presence of curcumin. Lipids rearrangements and drug partitioning caused changes of lipid fluidity. Such nonspecific effects of curcumin on cellular membranes would produce artifacts of apoptosis measurement, since several methods are based on membrane changes.     

Intracellular nanovesicles mediate α5β1 integrin trafficking during cell migration

Membrane traffic is an important regulator of cell migration through the endocytosis and recycling of cell surface receptors such as integrin heterodimers. Intracellular nanovesicles (INVs) are transport vesicles that are involved in multiple membrane trafficking steps, including the recycling pathway. The only known marker for INVs is tumor protein D54 (TPD54/TPD52L2), a member of the TPD52-like protein family. Overexpression of TPD52-like family proteins in cancer has been linked to poor prognosis and an aggressive metastatic phenotype, which suggests cell migration may be altered under these conditions. Here, we show that TPD54 directly binds membrane and associates with INVs via a conserved positively charged motif in its C terminus. We describe how other TPD52-like proteins are also associated with INVs, and we document the Rab GTPase complement of all INVs. Depletion of TPD52-like proteins inhibits cell migration and invasion, while their overexpression boosts motility. We show that inhibition of migration is likely due to altered recycling of α5β1 integrins in INVs.     

The proteomics of plant cell membranes

Membrane proteins are involved in many different functions depending on their location in the cell. Characterization of the membrane proteome can bring new insights to the function of different plant membrane systems and the subcellular compartments where the proteins are found. Plant membrane proteomics can also provide valuable information about plant-specific biological processes. Despite recent advances in the separation and techniques for the analysis of plant membrane proteins, characterization of these proteins, especially the hydrophobic ones, is still challenging. In this review, plant membrane proteomics data, compiled from the literature on Arabidopsis thaliana, are described. In addition, initial attempts towards determining the physiological significance of some proteins identified from membrane proteomics in rice are also described.     

Cross bonding and stiffening of the red cell membrane

Cross bonding and stiffening of the human red cell membrane was studied using treatments with SH, amino, and carboxyl reagents, oxidizing and denaturing treatments and acidification. Membrane cross bonding was initiated when, after red cell treatment, opposite areas of the cytoplasmic face of the red cell membrane were brought into contact by cell shrinking. Membrane cross bonding was detected by light microscopy when this contact persisted upon swelling the cells in a hypotonic medium. Membrane stiffening was recorded as a decrease in elongation of red cells in the shear field of a viscous dextran solution. No correlation was found between membrane cross bonding and membrane stiffening. The results are explained by the existence of two modifications of spectrin, type I causing solely membrane stiffening, type II causing membrane cross bonding as well as membrane stiffening. The amino and carboxyl reagents caused only type I modification. The other treatments caused both types of modification although with varying proportions. The results support the previously suggested mechanism of membrane cross bonding which involves a rearrangement of spectrin similar to denaturation by heat or urea, a decrease in associations within the membrane skeletal network, and a lateral aggregation of membrane proteins. These changes are proposed to occur by the type II modification. The data further substantiate the membrane stiffening effect of inter- and intra-molecular cross linking of spectrin which is identified with the type I modification. Finally, hypotheses are presented concerning the mechanism of membrane stiffening due to type II modifications of spectrin.     

Protruding membrane nanotubes: attachment of tubular protrusions to adjacent cells by several anchoring junctions

Membrane nanotubes are a morphologically versatile group of membrane structures (some resembling filopodia), usually connecting two closely positioned cells. In this article, we set morphological criteria that distinguish the membrane nanotubes from filopodia, as there is no specific molecular marker known to date that unequivocally differentiates between filopodia and protruding nanotubes. Membrane nanotubes have been extensively studied from the morphological point of view and the transport that can be conducted through them, but little is known about the way they connect to the adjacent cell. Our results show that the nanotubes may connect to a neighboring cell by anchoring junctions. Among cell adhesion proteins, N-cadherin, β-catenin, nectin-2, afadin and the desmosomal protein desmoplakin-2 were immune-labeled. We found that N-cadherin and β-catenin are concentrated in nanotubes, while the concentrations of other junction-involved proteins are not increased in these structures. On the basis of data from transmission electron microscopy, we propose a model of the nanotube attachment where the connection of nanotubes is stabilized by several anchoring junctions, most likely adherens junctions that are formed when the nanotube is sliding along the target cell membrane.     

中华眼镜蛇毒心脏毒素对人肝癌细胞株细胞膜的影响

目的和方法：运用５７０ 型粘附式细胞仪光漂白后荧光再分布法测定单个细胞膜脂流动性的动态变化和流式细胞仪测定细胞群细胞膜电位,以观察中华眼镜蛇毒心脏毒素对人肝癌细胞株细胞膜的影响。结果：心脏毒素使肝癌细胞膜脂流动性下降,并且使肝癌细胞膜电位下降。结论：心脏毒素对人肝癌细胞株Ｈ７４０２细胞膜有损伤作用     

Proteomics of total membranes and subcellular membranes

Membrane proteins are key molecules in the cell and are important targets for drug development. Much effort has, therefore, been directed towards research of this group of proteins, but their hydrophobic nature can make working with them challenging. Here we discuss methodologies used in the study of the membrane proteome, specifically discussing approaches that circumvent technical issues specific to the membrane. In addition, we review several techniques used for visualization, qualification, quantitation and localization of membrane proteins. The combination of the techniques we describe holds great promise to allow full characterization of the membrane proteome and to map the dynamic changes within it essential for cellular function.     

Proteomics of total membranes and subcellular membranes

Membrane proteins are key molecules in the cell and are important targets for drug development. Much effort has, therefore, been directed towards research of this group of proteins, but their hydrophobic nature can make working with them challenging. Here we discuss methodologies used in the study of the membrane proteome, specifically discussing approaches that circumvent technical issues specific to the membrane. In addition, we review several techniques used for visualization, qualification, quantitation and localization of membrane proteins. The combination of the techniques we describe holds great promise to allow full characterization of the membrane proteome and to map the dynamic changes within it essential for cellular function.     

Overcoming the challenges of membrane protein crystallography

Membrane protein structural biology is still a largely unconquered area, given that approximately 25% of all proteins are membrane proteins and yet less than 150 unique structures are available. Membrane proteins have proven to be difficult to study owing to their partially hydrophobic surfaces, flexibility and lack of stability. The field is now taking advantage of the high-throughput revolution in structural biology and methods are emerging for effective expression, solubilisation, purification and crystallisation of membrane proteins. These technical advances will lead to a rapid increase in the rate at which membrane protein structures are solved in the near future.