Proteomics of plasma membrane microdomains

Plasma membrane microdomains represent subcompartments of the plasma membrane characterized by a specific lipid and protein composition. The recognition of microdomains in nearly all the eukaryotic membranes has accredited them with specialized functions in health and disease. Several proteomic studies have recently addressed the specific composition of plasma membrane microdomains, and will be reviewed in this paper. Peculiar information has been obtained, but a comprehensive view of the main protein classes required to define the microdomain proteome is still missing. The achievement of this information is slowed by the difficulties encountered in resolving and analyzing hydrophobic proteins, but it could help in understanding the overall function of plasma membrane microdomains and their involvement in human pathology.     

Proteomic analysis of chlorosome-depleted membranes of the green sulfur bacterium Chlorobium tepidum

Green sulfur bacteria are obligate anaerobic phototrophs, which in addition to outer and plasma membranes contain chlorosomes. The analysis of the membrane proteome of Chlorobium tepidum from chlorosome-depleted membranes is described in this study. The membranes were purified by sucrose density centrifugation and characterized by 1-DE and 2-DE coupled with MS, absorption spectroscopy, and electron microscopy. 1-DE and 2-DE were employed to analyze the membrane proteins and to characterize the capabilities of the methods. Solubilization of the membrane proteins prior to 2-DE was improved by using a series of zwitterionic detergents. Based on the resolved spots after 2-DE, the combination of amidosulfobetaine 14 with Triton X-100 is more efficient than the combination of CHAPS, N-decyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, and Triton X-100. From the application of 1-DE and 2-DE, 167 and 202 unique proteins were identified, respectively, using PMF by MALDI-TOF MS. Both methods resulted in the detection of 291 different proteins of which only 88 were predicted membrane proteins, indicating the limitation of membrane protein detection after separation with electrophoresis methods. In addition, 53 of these proteins were identified as outer membrane proteins.     

Dynamics of plasma membrane microdomains and cross-talk to the insulin signalling cascade

The critical role of the heterogeneous nature of cellular plasma membranes in transmembrane signal transduction has become increasingly appreciated during the past decade. Areas of relatively disordered, loosely packed phospholipids are disrupted by hydrophobic detergent/carbonate-insoluble glycolipid-enriched raft microdomains (DIGs) of highly ordered (glyco)sphingolipids and cholesterol. DIGs exhibit low buoyant density and are often enriched in glycosylphosphatidylinositol-anchored plasma membrane proteins (GPI proteins), dually acylated signalling proteins, such as non-receptor tyrosine kinases (NRTKs), and caveolin. At least two types of DIGs, hcDIGs and lcDIGs, can be discriminated on basis of higher and lower content, respectively, of these typical DIGs components. In quiescent differentiated cells, GPI proteins and NRTKs are mainly associated with hcDIGs, however, in adipose cells certain insulin-mimetic stimuli trigger redistribution of subsets of GPI proteins and NRTKs from hcDIGs to lcDIGs. Presumably, these stimuli induce displacement of GPI proteins from a GPI receptor located at hcDIGs whereas simultaneously NRTKs dissociate from a complex with caveolin located at hcDIGs, too. NRTKs are thereby activated and, in turn, modulate intracellular signalling pathways, such as stimulation of metabolic insulin signalling in insulin-sensitive cells. The apparent dynamics of DIGs may provide a target mechanism for regulating the activity of lipid-modified signalling proteins by small drug molecules, as exemplified by the sulfonylurea, glimepiride, which lowers blood glucose in an insulin-independent fashion, in part.     

Proteomic analysis of the effect of Antrodia camphorata extract on human lung cancer A549 cell

Antrodia camphorata (niu-chang-chih) is a fungus native to Taiwan that is believed to be effective in preventing diseases. This study demonstrates that 0.2-2% v/v ethanol extracts of A. camphorata cultivated by solid-state fermentation (SACE) can effectively impede the proliferation of human non-small cell lung carcinoma A549 cells but not primary human fetal lung fibroblast MRC-5. The results of apoptotic analyses implicate that SACE might trigger the apoptosis in the A549 cells by inducing endoplasmic reticulum stress. Two-dimensional gel maps of non-treated and treated A549 cells were compared using PDQUEST analytical software to discover five statistically significant twofold or above-twofold differentially-expressed protein spots. The five protein spots that were significantly de-regulated were chosen for subsequent identification by high performance liquid chromatography electro-spray tandem mass spectrometry. The five proteins were later identified as human galectin-1, human eukaryotic translation initiation factor 5A, human Rho GDP dissociation inhibitor alpha, human calcium-dependent protease small subunit and human annexin V. All five proteins were confirmed to be down-regulated by Western blotting. The analytical results of this study help to provide insight into the effect of SACE on the gene expression of the tumor cells.     

ErbB-4 and TNF-alpha converting enzyme localization to membrane microdomains

Sequential proteolytic processing of ErbB-4 occurs in response to ligand addition. Here, we assess the localization of cleavable and non-cleavable ErbB-4 isoforms to membrane microdomains using three methodologies: (1) Triton X-100-insolubility, (2) Brij98-insolubility, and (3) detergent-free density gradient centrifugation. Whereas ErbB-4 translocated to a Triton X-100-insoluble fraction upon treatment of T47D cells with heregulin, it constitutively associated with a Brij98-insoluble fraction and a lipid raft fraction isolated using detergent-free methodology. Comparison of cleavable and non-cleavable isoforms of ErbB-4 revealed that both ErbB-4 isoforms are constitutively localized to either a Triton X-100-soluble or Brij98-insoluble fraction. In contrast, addition of heregulin resulted in translocation of the cleavable isoform to a detergent-free lipid raft. Tumor necrosis factor-alpha converting enzyme (TACE), the ectodomain secretase for ErbB-4, was present predominantly in its mature active form in most microdomains analyzed. These data suggest the assembly of ErbB-4 ectodomain cleavage apparatus in a membrane microdomain.     

Proteomic analysis of SET-binding proteins

The protein SET is involved in essential cell processes such as chromatin remodeling, apoptosis and cell cycle progression. It also plays a critical role in cell transformation and tumorogenesis. With the aim to study new SET functions we have developed a system to identify SET-binding proteins by combining affinity chromatography, MS, and functional studies. We prepared SET affinity chromatography columns by coupling the protein to activated Sepharose 4B. The proteins from mouse liver lysates that bind to the SET affinity columns were resolved with 2-DE and identified by MS using a MALDI-TOF. This experimental approach allowed the recognition of a number of SET-binding proteins which have been classified in functional clusters. The identification of four of these proteins (CK2, eIF2alpha, glycogen phosphorylase (GP), and TCP1-beta) was confirmed by Western blotting and their in vivo interactions with SET were demonstrated by immunoprecipitation. Functional experiments revealed that SET is a substrate of CK2 in vitro and that SET interacts with the active form of GP but not with its inactive form. These data confirm this proteomic approach as a useful tool for identifying new protein-protein interactions.     

Cellular aging-dependent decrease in cholesterol in membrane microdomains of human diploid fibroblasts

Recently it has been shown that cholesterol plays indispensable roles in the function of cholesterol-rich microdomains (rafts), such as in ligand-mediated signal transduction. Using a perfringolysin O derivative (BCtheta) that binds selectively to cholesterol in rafts without causing membrane damage (Proc. Natl. Acad. Sci. USA 98 (2001) 4926), we have investigated the effect of in vitro replicative aging of human diploid fibroblasts, TIG-1, on the distribution of plasma membrane cholesterol. The amount of BCtheta-labeled membrane cholesterol decreased during replicative aging of TIG-1 cells, whereas total cholesterol increased somewhat. The relationship was confirmed by double staining with BCtheta and senescence-associated-beta-galactosidase, a biomarker of senescent cells. Cell fractionation experiments revealed decreases in both cholesterol in rafts and a raft marker, flotillin, during replicative aging. In addition, hydroxyurea-induced prematurely senescent cells also showed a lower level of BCtheta-labeled cholesterol than untreated cells, despite maintaining the total amount of cholesterol. When TIG-1 cells were cultured in cholesterol-deficient medium, BCtheta labeling was first diminished and then premature senescence was induced. Taken together with the reduced signaling capacity of aged cells, these results suggest that plasma membrane cholesterol in raft microdomains is more sensitive to senescence than total cholesterol and is a primary target in aging.     

Proteomic analysis of membrane rafts of melanoma cells identifies protein patterns characteristic of the tumor progression stage

The molecular mechanisms controlling the progression of melanoma from a localized tumor to an invasive and metastatic disease are poorly understood. In the attempt to start defining a functional protein profile of melanoma progression, we have analyzed by LC-MS/MS the proteins associated with detergent resistant membranes (DRMs), which are enriched in cholesterol/sphingolipids-containing membrane rafts, of melanoma cell lines derived from tumors at different stages of progression. Since membrane rafts are involved in several biological processes, including signal transduction and protein trafficking, we hypothesized that the association of proteins with rafts can be regulated during melanoma development and affect protein function and disease progression. We have identified a total of 177 proteins in the DRMs of the cell lines examined. Among these, we have found groups of proteins preferentially associated with DRMs of either less malignant radial growth phase/vertical growth phase (VGP) cells, or aggressive VGP and metastatic cells suggesting that melanoma cells with different degrees of malignancy have different DRM profiles. Moreover, some proteins were found in DRMs of only some cell lines despite being expressed at similar levels in all the cell lines examined, suggesting the existence of mechanisms controlling their association with DRMs. We expect that understanding the mechanisms regulating DRM targeting and the activity of the proteins differentially associated with DRMs in relation to cell malignancy will help identify new molecular determinants of melanoma progression.     

Proteomic analysis of membrane proteins expressed specifically in pluripotent murine embryonic stem cells

Embryonic stem cells (ESCs) are established from the inner cell mass of preimplantation embryos, are capable of self‐renewal, and exhibit pluripotency. Given these unique properties, ESCs are expected to have therapeutic potential in regenerative medicine and as a powerful tool for in vitro differentiation studies of stem cells. Various growth factors and extracellular matrix components regulate the pluripotency and differentiation of ESC progenies. Thus, the cell surface receptors that bind these regulatory factors are crucial for the precise regulation of stem cells. To identify membrane proteins that are involved in the regulation of pluripotent stem cells, the membrane proteins of murine ESCs cultured with or without leukemia inhibitory factor (LIF) were purified and analyzed by quantitative proteomics. 2‐D PAGE‐based analysis using fluorescently labeled proteins and shotgun‐based analysis with isotope‐labeled peptides identified 338 proteins, including transmembrane, membrane‐binding, and extracellular proteins, which were expressed specifically in pluripotent or differentiated murine ESCs. Functions of the identified proteins revealed cell adhesion molecules, channels, and receptors, which are expected to play important roles in the maintenance of murine ESC pluripotency. Membrane proteins that are expressed in pluripotent ESCs but not in differentiated cells such as Slc16a1 and Bsg could be useful for the selection of the stem cells in vitro.     

Profiling of membrane proteins from human macrophages: comparison of two approaches

Macrophages are involved in various important biological processes and their functions are tightly regulated. Hydrophobic proteins are difficult to analyse by 2-DE because of their intrinsic tendency to self-aggregate during the first dimension (IEF). We have compared two protocols for extracting, separating and identifying membrane proteins from human macrophages by MALDI-TOF MS. The first protocol used protein extraction by solvent, followed by 2-DE and allowed us to identify 10% membrane proteins among the proteins identified a being like the peroxisome-activated receptor delta. The second method is based on solubilizing the membranes with Triton X-100, separating the proteins by anion-exchange chromatography followed by SDS-PAGE. This method allowed us to identify 49 membrane proteins, including four integral membrane proteins, ten type I, two type II and one type III membrane proteins. Several receptors were identified, including integrin alpha-3 and ephrin type A receptor 7. Interestingly, several proteins involved in macrophage functions were identified, such as integrin alpha-X and macrophage mannose receptor. These findings show that techniques are available to identify membrane proteins, but that they require large quantities of cells which means that they are not suitable for the limiting amounts of precious samples available from clinical studies.     

Insights into the membrane proteome of rat liver peroxisomes: microsomal glutathione-S-transferase is shared by both subcellular compartments

Peroxisomes are ubiquitous "multipurpose" organelles of eukaryotic cells. Their matrix enzymes catalyze mainly catabolic and anabolic reactions of lipid metabolism, thus contributing to the regulation of lipid homeostasis. Since most metabolites must be actively transported across the peroxisomal membrane and since individual proteins and protein complexes play functional roles in such transport processes, we analyzed the peroxisomal membrane proteome. Benzyldimethyl-n-hexadecylammoniumchloride (16-BAC)/SDS-2-D-PAGE and mass spectrometry were used to characterize the proteomes of highly purified "light" and "heavy" peroxisomes of rat liver obtained by density gradient centrifugation. In both populations, the major integral membrane proteins could be detected in high concentrations, verifying 16-BAC/SDS-2-D-PAGE as a suitable tool for the preparation of membrane proteomes destined for mass spectrometric analysis. Both reliable and reproducible detection of a distinct set of microsomal (ER) membrane proteins, including microsomal glutathione-S-transferase (mGST), in light and heavy peroxisomal fractions was also possible. Compared with the abundance of most microsomal membrane proteins, we found mGST to be specifically enriched in peroxisomal membrane fractions. Furthermore, C terminus epitope-tagged mGST versions were localized at least in part to peroxisomes in different mammalian cell lines. Taken together, these data suggest that the peroxisomal GST is not a mere ER-contaminant, but a bona fide protein comprising the membrane proteome of both intracellular compartments. In addition, we could detect several mitochondrial proteins in light peroxisome fractions. This finding may likely indicate a physical association of light peroxisomes with mitochondria, since the organelles could be partly separated by mechanical stress. Whether this association is of functional importance awaits further investigation.     

Use of Bodipy-labeled sphingolipid and cholesterol analogs to examine membrane microdomains in cells

Much evidence has accumulated to show that cellular membranes such as the plasma membrane, contain multiple "microdomains" of differing lipid and protein composition and function. These domains are sometimes enriched in cholesterol and sphingolipids and are believed to be important structures for the regulation of many biological and pathological processes. This review focuses on the use of fluorescent (Bodipy) labeled analogs of sphingolipids and cholesterol to study such domains. We discuss the similarities between the behavior of Bodipy-cholesterol and natural cholesterol in artificial bilayers and in cultured cells, and the use of Bodipy-sphingolipid analogs to visualize membrane domains in living cells based on the concentration-dependent monomer-excimer fluorescence properties of the Bodipy-fluorophore. The use of Bodipy-D-erythro-lactosylceramide is highlighted for detection of domains on the plasma membrane and endosome membranes, and the importance of the sphingolipid stereochemistry in modulating domain formation is discussed. Finally, we suggest that Bodipy-sphingolipids may be useful in future studies to examine the relationship between membrane domains at the cell surface and domains enriched in other lipids and proteins on the inner leaflet of the plasma membrane.     

Different glycoforms of the human GPI-anchored antigen CD52 associate differently with lipid microdomains in leukocytes and sperm membranes

CD52 is a human GPI-anchored antigen, expressed exclusively in the immune system and part of the reproductive system (epididymal cells). Sperm cells acquire the antigen from the epididymal secretions when transiting in the epididymal corpus and cauda. The peptide backbone of CD52, consisting of only 12 aminoacids, is generally considered no more than a scaffold for post-translational modifications, such as GPI-anchor and especially N-glycosylation which occur at the third asparagine. The latter modification is highly heterogeneous, especially in the reproductive system, giving rise to many different glycoforms, some of which are tissue specific. A peculiar O-glycan-containing glycoform is also found in reproductive and immune systems. We determined to locate CD52 in microdomains of leukocytes and sperm membranes using two antibodies: (1) CAMPATH-1G, the epitope of which includes the last three aminoacids and part of the GPI-anchor of glycoforms present in leukocytes and sperm cells; (2) anti-gp20, the epitope of which belongs to the unique O-glycan-bearing glycoform also present in both cell types. Using a Brij 98 solubilization protocol and sucrose gradient partition we demonstrated that the CD52 glycoforms recognized by both antibodies are markers of typical raft microdomains in leukocytes, whereas in capacitated sperm the O-glycoform is included in GM3-rich microdomains different from the cholesterol and GM1-rich lipid rafts with which CAMPATH antigen is stably associated. The importance of the association between GM3 and O-glycans for formation of specialized microdomains was confirmed by heterologous CD52 insertion experiments. When prostasomes from human seminal fluid were incubated with rat sperm from different epididymal regions, the CD52 glycoform recognized by anti-gp20 decorated rat epididymal corpus and cauda sperm, associated with the same low-cholesterol GM3-rich sperm membrane fractions as in human sperm. The glycoforms recognized by CAMPATH-1G were not found in rat sperm. The relationship between this differential insertion and differences in glycosylation of rat and human CD52 is discussed.     

Proteomic analysis of mouse liver plasma membrane: use of differential extraction to enrich hydrophobic membrane proteins

To comprehensively identify proteins of liver plasma membrane (PM), we isolated PMs from mouse liver by sucrose density gradient centrifugation. An optimized extraction method for whole PM proteins and several methods of differential extraction expected to enrich hydrophobic membrane proteins were tested. The extracted PM proteins were separated by 2-DE, and were identified by MALDI-TOF-MS, and ESI-quadrupole-TOF MS. As the complementary method, 1-DE-MS/MS was also used to identify PM proteins. The optimized lysis buffer containing urea, thiourea, CHAPS and NP-40 was able to extract more PM proteins, and treatment of PM samples with chloroform/methanol and sodium carbonate led to enrichment of more hydrophobic PM proteins. From the mouse liver PM fraction, 175 non-redundant gene products were identified, of which 88 (about 50%) were integral membrane proteins with one to seven transmembrane domains. The remaining products were probably membrane-associated and cytosolic proteins. The function distribution of all the identified liver PM proteins was analyzed; 40% represented enzymes, 12% receptors and 9% proteins with unknown function.     

Proteomic analysis of the cold stress response in the moss,Physcomitrella patens

Cold stress has adverse effects on plant growth and development. Plants respond and acclimate to cold stress through various biochemical and physiological processes, thereby acquiring stress tolerance. To better understand the basis for tolerance, we carried out a proteomic study in the model moss, Physcomitrella patens, characterizing gametophore proteins with 2‐DE and mass spectroscopy. Following exposure to 0°C for up to 3 days, out of the more than 1000 protein spots reproducibly resolved, only 45 changed in abundance by at least 1.5‐fold. Of these, 35 were identified by tryptic digestion and mass spectroscopy. Photosynthetic proteins decreased, whereas many catabolic proteins increased. In addition, cold stress up‐regulated a variety of signaling, cytoskeleton, and defense proteins and few proteins in these classes were down‐regulated. Up‐regulated proteins include the 14‐3‐3‐like protein, actin, HSP70s, lipoxygenases, and cytochrome P450 proteins. These results point to pathways that are important for the mechanism of cold stress response in P. patens and by extension to the entire plant kingdom.     

Priming-induced localization of G(ialpha2) in high density membrane microdomains

Subcellular fractionation of human neutrophils on linear sucrose density gradients was utilized to test the hypothesis that priming regulates the subcellular and sub-plasma membrane distribution of neutrophil G-protein subunits, G(ialpha2) and G(ialpha3), N-formyl peptide receptor, Lyn kinase and phospholipase C(beta2). G(ialpha2), but not G(ialpha3), moved from a lighter to a higher density plasma membrane fraction. Unoccupied N-formyl peptide receptors were found throughout the plasma membrane fractions and this distribution did not change with priming. In unprimed cells G(ialpha2) and its effector, phospholipase C(beta2), were segregated in different membrane compartments; priming caused G(ialpha2) to move to the compartment in which phospholipase C(beta2) resided. Thus, an important component of the mechanism of priming may involve regulation of the location of G-proteins and effector molecules in plasma membrane compartments where their abilities to couple may be enhanced.     

Proteomic analysis reveals perturbed energy metabolism and elevated oxidative stress in hearts of rats with inborn low aerobic capacity

Selection on running capacity has created rat phenotypes of high-capacity runners (HCRs) that have enhanced cardiac function and low-capacity runners (LCRs) that exhibit risk factors of metabolic syndrome. We analysed hearts of HCRs and LCRs from generation 22 of selection using DIGE and identified proteins from MS database searches. The running capacity of HCRs was six-fold greater than LCRs. DIGE resolved 957 spots and proteins were unambiguously identified in 369 spots. Protein expression profiling detected 67 statistically significant (p<0.05; false discovery rate <10%, calculated using q-values) differences between HCRs and LCRs. Hearts of HCR rats exhibited robust increases in the abundance of each enzyme of the β-oxidation pathway. In contrast, LCR hearts were characterised by the modulation of enzymes associated with ketone body or amino acid metabolism. LCRs also exhibited enhanced expression of antioxidant enzymes such as catalase and greater phosphorylation of α B-crystallin at serine 59, which is a common point of convergence in cardiac stress signalling. Thus, proteomic analysis revealed selection on low running capacity is associated with perturbations in cardiac energy metabolism and provided the first evidence that the LCR cardiac proteome is exposed to greater oxidative stress.     

Proteomic analysis of sera from hepatocellular carcinoma patients after radiofrequency ablation treatment

Comparative proteomic analysis was used to search for characteristic alterations in the sera of hepatocellular carcinoma (HCC) patients who had undergone curative radiofrequency ablation treatment. Serum samples collected from eight patients before and after treatment were subjected to 2-DE. Eighty-eight protein spots differentially expressed with the treatment were selected by clustering analysis, and the proteins were identified by MS based on MALDI-TOF/TOF analysis and public database searches. The statistical analysis suggested that four proteins decreased after treatment (pro-apolipoprotein, alpha2-HS glycoprotein, apolipoprotein A-IV precursor, and PRO1708/PRO2044, which is the carboxy terminal fragment of albumin) and that seven proteins were increased after treatment, including leucine-rich alpha2-glycoprotein and alpha1-antitrypsin. These data facilitate the identification of differentially expressed proteins that are involved in HCC carcinogenesis and provide candidate biomarkers for the development of diagnostic and therapeutic tools.