Colon cancer specific nuclear matrix protein alterations in human colonic adenomatous polyps

Most colon cancers arise within preexisting adenomatous polyps or adenomas. The slow evolution from the non-invasive premalignant lesion, the adenomatous polyp, to invasive cancer supports a strategy of early detection. Recently, we identified unique nuclear matrix proteins (NMPs) specific for colon cancer (CC2, CC3, CC4, CC5). Most of the NMPs identified are common to all cell types, but several identified NMPs are tissue and cell line specific. The objective of this study is to describe and characterize the NMP profile of premalignant adenomatous colon polyps. Specifically when in the adenoma-carcinoma sequence four specific colon cancer NMPs, previously described, appear. Using two-dimensional (2-D) gel analysis 20 colon polyps (one juvenile polyp, six tubular adenoma (TA), seven tubulovillous adenoma (TVA), six TVA with focal high-grade dysplasia (HGD), were analyzed for the presence of four (CC2, CC3, CC4, CC5) specific NMPs. CC2 was not seen in any of the premalignant polyps. CC5 was present in only two premalignant TVA with HGD and in one TA. CC3 and CC4 were present in most adenomas. None of the NMPs were seen in the juvenile polyp, which is not considered to be a precursor of colon cancer. CC2 and CC5 are NMPs expressed at the junction of an advanced adenoma and invasive colorectal cancer. CC3 and CC4 are expressed earlier in the evolution of adenomatous polyps. Development of an assay to these proteins may serve as a new method for early detection of colorectal cancer.     

Freeze-fracture analysis of phloem structure in plant tissue cultures. II. The sieve element plasma membrane

Sieve element plasma membranes reveal a unique distribution of intramembrane particles (IMPs) in tissue cultures fixed and cyroprotected prior to freeze-fracturing. Sieve element IMPs are smaller than those found in the plasma membranes of callus parenchyma cells from these same cultures. The PF/EF ratio of plasma membrane IMPs is 9.6 for parenchyma cells and 1.21 for sieve elements. The increased binding of IMPs to the sieve element E face may be related to the role of membrane proteins in the loading of sucrose and other molecules by these cells. The enlargement of the cell wall at the site of sieve area pores creates complementary ridges and depressions in the E and P fracture faces of sieve element plasma membranes. No alteration of IMP density is seen at the sieve area pore site.     

Scanning N-glycosylation mutagenesis of membrane proteins

N-Glycosylation of eukaryotic membrane proteins is a co-translational event that occurs in the lumen of the endoplasmic reticulum (ER). This process is catalyzed by a membrane-associated oligosaccharyl transferase (OST) complex that transfers a preformed oligosaccharide (Glc(3)Man(9)GlcNAc(2)-) to an asparagine (Asn) side-chain acceptor located within the sequon (-Asn-X-Ser/Thr-). Scanning N-glycosylation mutagenesis experiments, where novel acceptor sites are introduced at unique sites within membrane proteins, have shown that the acceptor sites must be located a minimum distance (12-14 amino acids) away from the luminal membrane surface of the ER in order to be efficiently N-glycosylated. Scanning N-glycosylation mutagenesis can therefore be used to determine membrane protein topology and it can also serve as a molecular ruler to define the ends of transmembrane (TM) segments. Furthermore, since N-glycosylation is a co-translational event, N-glycosylation mutagenesis can be used to identify folding intermediates in membrane proteins that may expose segments to the ER lumen transiently during biosynthesis.     

Triton X-114 phase separation in the isolation and purification of mouse liver microsomal membrane proteins

Integral membrane proteins (IMPs) mediate several cellular functions including cell adhesion, ion and nutrient transport, and cell signalling. IMPs are typically hard to isolate and purify due to their hydrophobic nature and low cellular abundance, however, microsomes are small lipid vesicles rich in IMPs, which form spontaneously when cells are mechanically disrupted. In this study, we have employed mouse liver microsomes as a model for optimising a method for IMP isolation and characterisation. Microsomes were collected by differential centrifugation, purified with sodium carbonate, and subjected to GeLC–MS/MS analysis. A total of 1124 proteins were identified in the microsome fraction, with 47% (524/1124) predicted by TMHMM to contain at least one transmembrane domain (TMD). The ability of phase partitioning using the detergent Triton X-114 (TX-114) to further enrich for membrane proteins was evaluated. Microsomes were subjected to successive rounds of solubility-based phase separation, with proteins partitioning into the aqueous phase, detergent phase, or TX-114-insoluble pellet fraction. GeLC–MS/MS analysis of the three TX-114 fractions identified 1212 proteins, of which 146 were not detected in the un-fractionated microsome sample. Conspicuously, IMPs partitioned to the detergent phase, with 56% (435/770) of proteins identified in that fraction containing at least one TMD. GO Slim characterisation of the microsome proteome revealed enrichment of proteins from the endoplasmic reticulum, mitochondria, Golgi apparatus, endosome, and cytoplasm. Further, enzymes including monooxygenases were well represented with 35 cytochrome P450 identifications (CYPs 1A2, 2A5, 2A12, 2B10, 2C29, 2C37, 2C39, 2C44, 2C50, 2C54. 2C67, 2C68, 2C70, 2D10, 2D11, 2D22, 2D26, 2D9, 2E1, 2F2, 2J5, 2U1, 3A11, 3A13, 3A25, 4A10, 4A12A, 4A12B, 4F13, 4F14, 4F15, 4V3, 51,7B1, and 8B1). Evaluation of biological processes showed enrichment of proteins involved in fatty acid biosynthesis and elongation, as well as steroid synthesis. In addition, transport proteins including 24 members of the Rab family of GTPases were identified. Comparison of this dataset with the current mouse liver microsome proteome contributes an additional 648 protein identifications, of which 50% (326/648) contain at least one TMD.     

N-糖基化在肿瘤细胞逃逸CD8+T细胞中的意义和机制

肿瘤在发生发展过程中会产生异常糖基化结构,改变细胞功能,使其能逃逸机体细胞的免疫识别和免疫攻击,实现免疫逃逸。细胞膜表面蛋白的N-糖基化参与调控肿瘤细胞的增殖、迁移、侵袭、干细胞特性等。近年来,一系列研究进一步证实,肿瘤细胞免疫检查点分子的N-糖基化,以及识别N-糖基化的动物凝集素在肿瘤免疫逃逸CD8⁺T细胞中发挥了重要作用,如PD-L1的N-糖基化能增强其自身蛋白质的稳定性,促进与其受体PD-1的相互作用,并且靶向PD-1、PD-L1蛋白N-糖基化的抗体,能够有效抑制肿瘤免疫逃逸和肿瘤发展。     

Quality control of integral membrane proteins

Integral membrane proteins (IMPs) are essential components of the plasma and organellar membranes of the eukaryotic cell. Non-native IMPs, which can arise as a result of mutations, errors during biosynthesis or cellular stress, can disrupt these membranes and potentially lead to cell death. To protect against this outcome, the cell possesses quality control (QC) systems that detect and dispose of non-native IMPs from cellular membranes. Recent studies suggest that recognition of non-native IMPs by the QC machinery is correlated with the thermodynamic stability of these proteins. Consistent with this, small molecules known as chemical and pharmacological chaperones have been identified that stabilize non-native IMPs and enable them to evade QC. These findings have far-reaching implications for treating human diseases caused by defective IMPs.     

Changes of nuclear matrix proteins following the differentiation of human osteosarcoma MG-63 cells

Human osteosarcoma MG-63 cells were induced into differentiation by 5 mmol/L hexamethylene bisacetamide （HMBA）. Their nuclear matrix proteins （NMPs） were selectively extracted and subjected to two-dimensional gel electrophoresis analysis. The results of protein patterns were analyzed by Melanie software. The spots of differentially expressed NMPs were excised and subjected to in situ digestion with trypsin. The maps of peptide mass fingerprinting were obtained by MALDI-TOF-MS analysis, and were submitted for NCBI database searches by Mascot tool. There were twelve spots changed remarkably during the differentiation induced by HMBA, nine of which were identified. The roles of the regulated proteins during the MG-63 differentiation were analyzed. This study suggests that the induced differentiation of cancer cells is accompanied by the changes of NMPs, and confirms the presence of some specific NMPs related to the cancer cell proliferation and differentiation. The changed NMPs are potential markers for cancer diagnosis or targets for cancer therapy.     

Complex N-Glycans Influence the Spatial Arrangement of Voltage Gated Potassium Channels in Membranes of Neuronal-Derived Cells

The intrinsic electrical properties of a neuron depend on expression of voltage gated potassium (Kv) channel isoforms, as well as their distribution and density in the plasma membrane. Recently, we showed that N-glycosylation site occupancy of Kv3.1b modulated its placement in the cell body and neurites of a neuronal-derived cell line, B35 neuroblastoma cells. To extrapolate this mechanism to other N-glycosylated Kv channels, we evaluated the impact of N-glycosylation occupancy of Kv3.1a and Kv1.1 channels. Western blots revealed that wild type Kv3.1a and Kv1.1 α-subunits had complex and oligomannose N-glycans, respectively, and that abolishment of the N-glycosylation site(s) generated Kv proteins without N-glycans. Total internal reflection fluorescence microscopy images revealed that N-glycans of Kv3.1a contributed to its placement in the cell membrane while N-glycans had no effect on the distribution of Kv1.1. Based on particle analysis of EGFP-Kv proteins in the adhered membrane, glycosylated forms of Kv3.1a, Kv1.1, and Kv3.1b had differences in the number, size or density of Kv protein clusters in the cell membrane of neurites and cell body of B35 cells. Differences were also observed between the unglycosylated forms of the Kv proteins. Cell dissociation assays revealed that cell-cell adhesion was increased by the presence of complex N-glycans of Kv3.1a, like Kv3.1b, whereas cell adhesion was similar in the oligomannose and unglycosylated Kv1.1 subunit containing B35 cells. Our findings provide direct evidence that N-glycans of Kv3.1 splice variants contribute to the placement of these glycoproteins in the plasma membrane of neuronal-derived cells while those of Kv1.1 were absent. Further when the cell membrane distribution of the Kv channel was modified by N-glycans then the cell-cell adhesion properties were altered. Our study demonstrates that N-glycosylation of Kv3.1a, like Kv3.1b, provides a mechanism for the distribution of these proteins to the cell body and outgrowths and thereby can generate different voltage-dependent conductances in these membranes.     

Improvement of hydrophobic integral membrane protein identification by mild performic acid oxidation-assisted digestion

Integral membrane proteins (IMPs) are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of IMPs make them difficult to analyze. In proteomic analyses, hydrophobic peptides including transmembrane domains are often underrepresented, and this reduces the sequence coverage and reliability of the identified IMPs. Here we report a new strategy, mild performic acid oxidation treatment (mPAOT), for improvement of IMP identification. In the mPAOT strategy, the hydrophobicity of IMPs is significantly decreased by oxidizing their methionine and cysteine residues with performic acid, thereby improving the solubility and enzymolysis of these proteins. The application of the mPAOT strategy to the analysis of IMPs from human nasopharyngeal carcinoma CNE1 cell line demonstrated that many IMPs, including those with high hydrophobicity, could be reliably identified.     

Recycling Endosome Membrane Incorporation into the Leading Edge Regulates Lamellipodia Formation and Macrophage Migration

In comparison to our knowledge of the recycling of adhesion receptors and actin assembly, exactly how the cell controls its surface membrane to form a lamellipodium during migration is poorly understood. Here, we show the recycling endosome membrane is incorporated into the leading edge of a migrating cell to expand lamellipodia membrane. We have identified the SNARE complex that is necessary for fusion of the recycling endosome with the cell surface, as consisting of the R‐SNARE VAMP3 on the recycling endosome partnering with the surface Q‐SNARE Stx4/SNAP23, which was found to translocate and accumulate on the leading edge of migrating cells. Increasing VAMP3‐mediated fusion of the recycling endosome with the surface increased membrane ruffling, while inhibition of VAMP3‐mediated fusion showed that incorporation of the recycling endosome is necessary for efficient lamellipodia formation. At the same time, insertion of this recycling endosome membrane also delivers its cargo integrin α5β1 to the cell surface. The loss of this extra membrane for lamellipodia expansion and delivery of cargo in cells resulted in macrophages with a diminished capacity to effectively migrate. Thus, the recycling endosome membrane is incorporated into the leading edge and this aids expansion of the lamellipodia and simultaneously delivers integrins necessary for efficient cell migration.     

Membrane differentiations of an absorptive epithelium covering embryonic trophotaeniae in a goodeid teleost

Summary The trophotaenial absorptive cells (TACs) in goodeid embryos facilitate nutrient absorption during prolonged periods of intraovarian gestation. In a study of membrane differentiations associated with solute and ligand transfer in the trophotaeniae of Xenotoca eiseni, embryos were incubated in vivo with cationized ferritin (CF) prior to freeze-cleaving. This exposure to high concentrations of an adsorptive ligand was meant to induce swelling of the endosomal compartment. Macromolecular trafficking in TACs occurs via an apical endocytic complex consisting of plasma membrane invaginations, a large population of small vesicles, uniformly thick apical tubules, and endosomes. Freeze-fracture replicas showed that the microvillar plasma membrane P-face of TACs was studded with intramembrane particles (IMPs) at a fairly high density, whereas that of the cell surface proper contained a distinctly lower density and the tubulovesicular endocytic pits contained almost no IMPs. The majority of small vesicles and apical tubules in a near surface position displayed P-fracture faces with only a few odd IMPs, indicating that membrane, shuttling between the apical plasma membrane and intracellular sorting organelles, obviously does not carry along many large-sized integral membrane proteins. The distended endosomal compartment had many P-face-associated particles primarily clustered into patches. Specializations of the lateral plasma membrane included 4–8 tight junctional strands, relatively large complements of gap junction proteins, and numerous plaques of desmosomal membrane particles. A system of lamellar cisternae underlay the lateral cell surface that was in continuity with the intraepithelial space by numerous tubular canals, giving rise to an intracellular amplification of the basolateral plasma membrane. Their outward openings appeared as tiny pits on the cytoplasmic faces of freeze-cleaved cell membrane. The density of IMPs on the P-faces of the surface plasma membrane was apparently lower than that on its invaginated lamellar complex. Hence, it is concluded that the mobility of integral membrane proteins in the plane of the membrane may be hampered in movement across the surface pores.Supported by the Deutsche Forschungsgemeinschaft (Schi 268/1-1)     

Membrane subproteomic analysis of Mycobacterium bovis bacillus Calmette-Guérin

Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been known for a long time to prevent tuberculosis (TB) worldwide since 1921. Nonetheless, we know little about BCG membrane proteome. In the present study, we utilized alkaline incubation and Triton X-114-based methods to enrich BCG membrane proteins and subsequently digested them using proteolytic enzyme. The recovered peptides were further separated by 2-D LC and identified by ESI-MS/MS. As a result, total 474 proteins were identified, including 78 integral membrane proteins (IMPs). Notably, 18 BCG IMPs were described for the first time in mycobacterium. Further analysis of the 78 IMPs indicated that the theoretical molecular mass distribution of them ranged from 8.06 to 167.86 kDa and pI scores ranged from 4.40 to 11.60. Functional classification revealed that a large proportion of the identified IMPs (67.9%, 53 out of 78) were involved in cell wall and cell processes functional group. In conclusion, here we reported a comprehensive profile of the BCG membrane subproteome. The present investigation may allow the identification of some valuable vaccine and drug target candidates and thus provide basement for future designing of preventive, diagnostic, and therapeutic strategies against TB.     

A combination of immobilised pH gradients improves membrane proteomics

Membrane protein analyses have been notoriously difficult due to hydrophobicity and the general low abundance of these proteins compared to their soluble cytosolic counterparts. Shotgun proteomics has become the preferred method for analyses of membrane proteins, in particular the recent development of peptide immobilized pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two-dimensional shotgun proteomics. Recently, peptide IPG-IEF has been shown to be a valuable shotgun proteomics technique through the use of acidic narrow range IPG strips, which demonstrated that small acidic p I increments are rich in peptides. In this study, we assess the utility of both broad range (BR) (p I 3-10) and narrow range (NR) (p I 3.4-4.9) IPG strips for rat liver membrane protein analyses. Furthermore, the use of these IPG strips was evaluated using label-free quantitation to demonstrate that the identification of a subset of proteins can be improved using NR IPG strips. NR IPG strips provided 2603 protein assignments on average (with 826 integral membrane proteins (IMPs)) compared to BR IPG strips, which provided 2021 protein assignments on average (with 712 IMPs). Nonredundant protein analysis demonstrated that in total from all experiments, 4195 proteins (with 1301 IMPs) could be identified with 1428 of these proteins unique to NR IPG strips with only 636 from BR IPG strips. With the use of label-free quantitation methods, 1659 proteins were used for quantitative comparison of which 319 demonstrated statistically significant increases in normalized spectral abundance factors (NSAF) in NR IPG strips compared to 364 in BR IPG strips. In particular, a selection of six highly hydrophobic transmembrane proteins was observed to increase in NSAF using NR IPG strips. These results provide evidence for the use of alternative pH gradients in combination to improve the shotgun proteomic analysis of the membrane proteome.     

VAMP8-dependent fusion of recycling endosomes with the plasma membrane facilitates T lymphocyte cytotoxicity

Cytotoxic T lymphocytes (CTLs) eliminate infected and neoplastic cells through directed release of cytotoxic granule contents. Although multiple SNARE proteins have been implicated in cytotoxic granule exocytosis, the role of vesicular SNARE proteins, i.e., vesicle-associated membrane proteins (VAMPs), remains enigmatic. VAMP8 was posited to represent the cytotoxic granule vesicular SNARE protein mediating exocytosis in mice. In primary human CTLs, however, VAMP8 colocalized with Rab11a-positive recycling endosomes. Upon stimulation, these endosomes rapidly trafficked to and fused with the plasma membrane, preceding fusion of cytotoxic granules. Knockdown of VAMP8 blocked both recycling endosome and cytotoxic granule fusion at immune synapses, without affecting activating signaling. Mechanistically, VAMP8-dependent recycling endosomes deposited syntaxin-11 at immune synapses, facilitating assembly of plasma membrane SNARE complexes for cytotoxic granule fusion. Hence, cytotoxic granule exocytosis is a sequential, multivesicle fusion process requiring VAMP8-mediated recycling endosome fusion before cytotoxic granule fusion. Our findings imply that secretory granule exocytosis pathways in other cell types may also be more complex than previously appreciated.     

Flexibility in targeting and insertion during bacterial membrane protein biogenesis

The biogenesis of Escherichia coli inner membrane proteins (IMPs) is assisted by targeting and insertion factors such as the signal recognition particle (SRP), the Sec-translocon and YidC with translocation of (large) periplasmic domains energized by SecA and the proton motive force (pmf). The use of these factors and forces is probably primarily determined by specific structural features of an IMP. To analyze these features we have engineered a set of model IMPs based on endogenous E. coli IMPs known to follow distinct targeting and insertion pathways. The modified model IMPs were analyzed for altered routing using an in vivo protease mapping approach. The data suggest a facultative use of different combinations of factors.     

From complementarity to comprehensiveness--targeting the membrane proteome of growing Bacillus subtilis by divergent approaches

The analysis of integral membrane proteins (IMPs) with mass spectrometry-centered technologies has undergone great progress during the past few years, allowing for the analysis of several hundreds of IMPs. In this study, we investigated three promising shotgun approaches for the identification of IMPs of the model organism Bacillus subtilis. One comprises a classical membrane preparation procedure with carbonate and high-ionic-strength buffers, followed by SDS-PAGE and LC-MS/MS analysis. The two others are based on enzymatic trimming of the crude membrane fraction either with trypsin or proteinase K and subsequent gel-free analysis. As a result, we observed the highest degree of complementarity between the gel-based and the proteinase K approach, since the first exclusively addresses soluble loops and domains of IMPs and gave rise to 8709 unique peptides, whereas the latter contributed 1180 unique peptide identifications from otherwise inaccessible transmembrane helices (TMHs). All three methods contribute significant numbers (381, 284, and 276, respectively) to the total of 527 IMP identifications from the membrane fraction of exponentially growing B. subtilis cells, thus representing approximately 69% of all transcribed IMPs.     

Membrane modifications in chick osteoclasts revealed by freeze-fracture replicas

Remarkable differences among various membranes of bone cells became evident by examination of freeze-fracture replicas. In osteoclasts, three types of intramembranous particles (IMPs) were identified based on their size and shape: two sizes of isolated globular particles (8 and 12 nm in diameter) and rod-shaped, linear aggregates (8 x 30 nm in dimension). Furthermore, the density and distribution pattern of these IMPs enabled us to distinguish three different domains of membranes of osteoclasts including ruffled border, clear zone, and basolateral regions, as were also observed in thin sections. The highest density of IMPs was 3,500-4,000/microns2 in the ruffled border membrane, and these IMPs included linear aggregates among the usual globular particles. Linear aggregated particles were also observed in the membrane of cytoplasmic vesicles in the vicinity of the ruffled border region, but not in this membrane in other bone cells. In attached osteoclasts, the distribution patterns and densities of IMPs in each ruffled-finger and -plate were extremely variable, from closely to the loosely packed membrane particles. Focal aggregates of membrane particles were also frequently encountered. An important outcome of the present study was the finding that the presence of linear aggregated particles proved to be an additional criterion for distinguishing membrane domains in freeze-replicas of osteoclasts. The surface of the clear zone membrane was not smooth in profile, but revealed a number of eminences that were almost free of particles. Basolateral membranes exhibited a particle density of 2,400/microns2. Globular particles were homogeneously scattered in random fashion on their exposed fracture faces. In some cases, aggregates of IMPs on the basolateral membranes were encountered. In comparison with the ruffled fingers, microprojections from the basolateral surface showed a lesser density of IMPs and were devoid of rod-shaped or linear aggregated particles. Differences between osteoblasts and osteocytes were apparent in the density and the size of IMPs. The membranes of osteoblasts and osteocytes contained the same types of globular particles as seen in osteoclasts. Various sizes of gap junctions were located only on basolateral membranes of the osteoblasts. In contrast, no cellular junctions were observed between osteoclasts and any other type of cells.     

Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages

Human immunodeficiency virus type 1 (HIV-1) infects CD4(+) T lymphocytes and monocytes/macrophages, incorporating host proteins in the process of assembly and budding. Analysis of the host cell proteins incorporated into virions can provide insights into viral biology. We characterized proteins in highly purified HIV-1 virions produced from human monocyte-derived macrophages (MDM), within which virus buds predominantly into intracytoplasmic vesicles, in contrast to the plasmalemmal budding of HIV-1 typically seen with infected T cells. Liquid chromatography-linked tandem mass spectrometry of highly purified virions identified many cellular proteins, including 33 previously described proteins in HIV-1 preparations from other cell types. Proteins involved in many different cellular structures and functions were present, including those from the cytoskeleton, adhesion, signaling, intracellular trafficking, chaperone, metabolic, ubiquitin/proteasomal, and immune response systems. We also identified annexins, annexin-binding proteins, Rab proteins, and other proteins involved in membrane organization, vesicular trafficking, and late endosomal function, as well as apolipoprotein E, which participates in cholesterol transport, immunoregulation, and modulation of cell growth and differentiation. Several tetraspanins, markers of the late endosomal compartment, were also identified. MDM-derived HIV contained 26 of 37 proteins previously found in exosomes, consistent with the idea that HIV uses the late endosome/multivesicular body pathway during virion budding from macrophages.     

Nuclear matrix proteins in well and poorly differentiated human breast cancer cell lines

The nuclear matrix, besides providing the structural support of the nucleus, is involved in various cellular functions of the nucleus. Nuclear matrix proteins (NMPs), which are both tissue- and cell type–specific, are altered with transformation and state of differentiation. Furthermore, NMPs have been identified as informative markers of disease states. Here, the NMP profiles from human breast cancer cell lines and breast tumours were analyzed using two-dimension gel electrophoresis. We identified NMPs that are associated with well and poorly differentiated human breast cancer cells in vitro and in vivo. Five NMPs (NMBC 1–5) were found to be exclusive for well-differentiated human breast cancer cells, while one NMP (NMBC-6) was found to be present only in poorly differentiated human breast cancer cells. The identification of these proteins suggests the potential use of nuclear matrix proteins as prognostic indicators. J. Cell. Biochem. 66:9–15, 1997. © 1997 Wiley-Liss, Inc.