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91.
Plasma membranes form a critical biological interface between the inside of every cell and its external environment. Their roles in multiple key cellular functions make them important drug targets. However the protein composition of plasma membranes in general is poorly defined as the inherent properties of lipid embedded proteins, such as their hydrophobicity, low abundance, poor solubility and resistance to digestion and extraction makes them difficult to isolate, solubilize, and identify on a large scale by traditional mass spectrometry methods. Here we describe some of the significant advances that have occurred over the past ten years to address these challenges including: i) the development of new and improved membrane isolation techniques via either subfractionation or direct labeling and isolation of plasma membranes from cells and tissues; ii) modification of mass spectrometry methods to adapt to the hydrophobic nature of membrane proteins and peptides; iii) improvements to digestion protocols to compensate for the shortage of trypsin cleavage sites in lipid-embedded proteins, particularly multi-spanning proteins, and iv) the development of numerous bioinformatics tools which allow not only the identification and quantification of proteins, but also the prediction of membrane protein topology, membrane post-translational modifications and subcellular localization. This review emphasis the importance and difficulty of defining cells in proper patho- and physiological context to maintain the in vivo reality. We focus on how key technological challenges associated with the isolation and identification of cell surface proteins in tissues using mass spectrometry are being addressed in order to identify and quantify a comprehensive plasma membrane for drug and target discovery efforts.Plasma membranes (PM)1 and their associated proteins are part of a key biological interface between the outside and the inside of the cell. They are implicated in important cellular functions, such as small molecule transport, cell communication, and signaling. Such proteins are critical in sensing changes in the external environment and in transmitting signals into and out of the cell. Impaired cellular signaling, often involving PM proteins, is apparent in many cancers (1, 2). Membrane proteins represent one-third of the proteins encoded by the human genome (3) but represent more than two-thirds of the known protein targets for existing drugs (4). Thus, defining the proteome of PMs is critical for understanding cellular functions and fundamental biological processes and for finding new targets for drug discovery efforts.In an ideal world, the PM proteome of all cell types would be analyzed comprehensively to (i) better understand why and where different membrane proteins are expressed, (ii) reveal new functions for the PM in various cell types, (iii) identify cell-specific surface-accessible markers as targetable proteins for local drug delivery, and (iv) identify diagnostic or prognostic indicators in healthy and diseased cell or tissue states. This can be attempted relatively easily in cell culture using a homogenous cell population as compared with the heterogeneity of cells within any tissue or organ from which they are derived. Unfortunately, once cells are isolated from different organs and even more so when cultured and grown in vitro, the cells can change dramatically in appearance, structure, and responsiveness, and protein expression and distribution within the cell can change (5, 6). Biomarkers readily disappear, and the cultured cells do not reflect the in vivo reality. Most importantly, they lose expression of tissue-specific proteins and dedifferentiate into a more common phenotype. Mass spectrometry (MS) analysis revealed that as much as 40% of the proteins expressed by endothelial cells in vivo are not found in vitro (6). Thus, several groups have tried to address this problem by capturing the PMs in as close to an in vivo situation as possible as discussed below.PM proteins in general may not serve as suitable targets for drug treatment regimens mainly because of their inaccessibility to intravenously injected agents. Although most small molecule drugs can readily penetrate and accumulate in almost any tissue, often a high dose must be administered for the therapeutic dosage to accumulate in the diseased tissue of interest. However, major problems can arise when such drugs are toxic to both normal and healthy tissue, e.g. chemotherapeutic agents, which can accumulate in healthy tissue, resulting in the unwanted and often severe side effects associated with these drugs. Thus, even if a protein expressed on the surface of a cell is indicative of a disease state, it would be relatively impossible to target this cell specifically while avoiding the accumulation in healthy tissue using current drug delivery approaches, especially if the cell of interest is embedded within a tissue or organ. This is simply because multiple barriers, e.g. endothelium, epithelium, etc., must be crossed to access the cell regardless of the route of administration. As a result, great effort is now being focused on developing more targeted approaches where the toxic agents are specifically delivered to the organ or tissue of interest (7–9). With such targeting approaches, dosages can be significantly reduced, thus increasing the therapeutic efficacy while minimizing the side effects (10, 11). Consequently, proteins that are expressed on the surface of endothelial cells (ECs) that line the luminal surface of all vasculature are attractive targets for drugs and imaging agents as these proteins are in direct contact with the circulating blood and are thus inherently more accessible to intravenously administered agents than PM proteins of cells residing deep inside tissues and organs. Proteins expressed on the outer luminal EC surface can readily bind antibodies and other agents that are circulating in the blood. Thus, identifying and characterizing the proteins that line the vasculature of each organ and tissue is highly desirable for drug delivery and diagnostic imaging.PM proteins, regardless of the cell or tissue of origin, have generally been under-represented in proteomics analysis mainly because of their low abundance. In addition, the inherent insolubility of membrane proteins due to their hydrophobic nature has rendered them difficult to isolate and identify compared with their counterparts in the soluble cytosol and nuclear fractions. In many high throughput protein identification approaches, soluble proteomes are readily characterized, and it is fairly common for thousands of proteins to be identified in such samples. However, when more challenging proteomes, such as those of the PM, are of interest, the numbers of proteins identified are significantly lower. There are many technical reasons for such a dramatic decrease in protein identification when membrane proteomes are of interest. This review focuses on how we and other laboratories are overcoming key technological challenges associated with using traditional MS-based approaches, which were initially developed for the identification of more soluble proteins, for the mapping of membrane proteomes and in particular the in vivo cell surface proteome of endothelial cells. 相似文献
92.
Maria Takacs Maxim V. Petoukhov R. Andrew Atkinson Pierre Roblin Fran?ois-Xavier Ogi Borries Demeler Noelle Potier Yassmine Chebaro Annick Dejaegere Dmitri I. Svergun Dino Moras Isabelle M. L. Billas 《PloS one》2013,8(7)
Background
PGC-1α is a crucial regulator of cellular metabolism and energy homeostasis that functionally acts together with the estrogen-related receptors (ERRα and ERRγ) in the regulation of mitochondrial and metabolic gene networks. Dimerization of the ERRs is a pre-requisite for interactions with PGC-1α and other coactivators, eventually leading to transactivation. It was suggested recently (Devarakonda et al) that PGC-1α binds in a strikingly different manner to ERRγ ligand-binding domains (LBDs) compared to its mode of binding to ERRα and other nuclear receptors (NRs), where it interacts directly with the two ERRγ homodimer subunits.Methods/Principal Findings
Here, we show that PGC-1α receptor interacting domain (RID) binds in an almost identical manner to ERRα and ERRγ homodimers. Microscale thermophoresis demonstrated that the interactions between PGC-1α RID and ERR LBDs involve a single receptor subunit through high-affinity, ERR-specific L3 and low-affinity L2 interactions. NMR studies further defined the limits of PGC-1α RID that interacts with ERRs. Consistent with these findings, the solution structures of PGC-1α/ERRα LBDs and PGC-1α/ERRγ LBDs complexes share an identical architecture with an asymmetric binding of PGC-1α to homodimeric ERR.Conclusions/Significance
These studies provide the molecular determinants for the specificity of interactions between PGC-1α and the ERRs, whereby negative cooperativity prevails in the binding of the coactivators to these receptors. Our work indicates that allosteric regulation may be a general mechanism controlling the binding of the coactivators to homodimers. 相似文献93.
Greenstein AE Echols N Lombana TN King DS Alber T 《The Journal of biological chemistry》2007,282(15):11427-11435
To define how extracellular signals activate bacterial receptor Ser/Thr protein kinases, we characterized the regulatory functions of a weak dimer interface identified in the Mycobacterium tuberculosis PknB and PknE receptor kinases. Sequence comparisons revealed that the analogous interface is conserved in PknD orthologs from diverse bacterial species. To analyze the roles of dimerization, we constructed M. tuberculosis PknD kinase domain (KD) fusion proteins that formed dimers upon addition of rapamycin. Dimerization of unphosphorylated M. tuberculosis PknD KD fusions stimulated phosphorylation activity. Mutations in the dimer interface reduced this activation, limited autophosphorylation, and altered substrate specificity. In contrast, an inactive catalytic site mutant retained the ability to stimulate the wild-type KD by dimerization. These results support the idea that dimer formation allosterically activates unphosphorylated PknD. The phosphorylated PknD KD was fully active even in the absence of dimerization, suggesting that phosphorylation provides an additional regulatory mechanism. The conservation of analogous dimers in diverse prokaryotic and eukaryotic Ser/Thr protein kinases implies that this mechanism of protein kinase regulation is ancient and broadly distributed. 相似文献
94.
Chae K Zhang K Zhang L Morikis D Kim ST Mollet JC de la Rosa N Tan K Lord EM 《The Journal of biological chemistry》2007,282(46):33845-33858
Lily pollen tubes grow adhering to an extracellular matrix produced by the transmitting tract epidermis in a hollow style. SCA, a small ( approximately 9.4 kDa), basic protein plus low esterified pectin from this extracellular matrix are involved in the pollen tube adhesion event. The mode of action for this adhesion event is unknown. We partially separated three SCA isoforms from the lily stigma in serial size exclusion column fractions (SCA1, 9370 Da; SCA2, 9384 Da; SCA3, 9484 Da). Peptide sequencing analysis allowed us to determine two amino acid variations in SCA3, compared with SCA1. For SCA2, however, there are more sequence variations yet to be identified. Our structural homology and molecular dynamics modeling results show that SCA isoforms have the plant nonspecific lipid transfer protein-like structure: a globular shape of the orthogonal 4-helix bundle architecture, four disulfide bonds, an internal hydrophobic and solvent-inaccessible cavity, and a long C-terminal tail. The Ala(71) in SCA3, replacing the Gly(71) in SCA1, has no predictable effect on structure. The Arg(26) in SCA3, replacing the Gly(26) in SCA1, is predicted to cause structural changes that result in a significantly reduced volume for the internal hydrophobic cavity in SCA3. The volume of the internal cavity fluctuates slightly during the molecular dynamics simulation, but overall, SCA1 displays a larger cavity than SCA3. SCA1 displays higher activity than SCA3 in the in vitro pollen tube adhesion assay. No differences were found between the two SCAs in a binding assay with pectin. The larger size of the hydrophobic cavity in SCA1 correlates with its higher adhesion activity. 相似文献
95.
96.
Pyridoxine is used as a supplement for treating conditions such as vitamin deficiency as well as neurological disorders such as depression, epilepsy and autism. A significant neurologic complication of pyridoxine therapy is peripheral neuropathy thought to be a result of long-term and high dose usage. Although pyridoxine-induced neuropathy is transient and can remit after its withdrawal, the process of complete recovery can be slow. Glutamate carboxypeptidase II (GCP II) inhibition has been shown to improve symptoms of both chemotherapy- and diabetic-induced neuropathy. This study evaluated if GCP II inhibition could behaviorally and physiologically improve pyridoxine-induced neuropathy. In the current study, high doses of pyridoxine (400 mg/kg, twice a day for seven days) were used to induce neuropathy in rats. An orally bioavailable GCP II inhibitor, 2-(3-mercaptopropyl) pentanedioic acid (2-MPPA), was administered daily at a dose of 30 mg/kg starting from the onset of pyridoxine injections. Body weight, motor coordination, heat sensitivity, electromyographical (EMG) parameters and nerve morphological features were monitored. The results show beneficial effects of GCP II inhibition including normalization of hot plate reaction time, foot fault improvements and increased open field distance travelled. H wave frequency, amplitude and latency as well as sensory nerve conduction velocity (SNCV) were also significantly improved by 2-MPPA. Lastly, GCP II inhibition resulted in morphological protection in the spinal cord and sensory fibers in the lumbar region dorsal root ganglia (DRG). In conclusion, inhibition of GCP II may be beneficial against the peripheral sensory neuropathy caused by pyridoxine. 相似文献
97.
The Protein Kinase G, EGL-4, is required within the C. elegans AWC sensory neurons to promote olfactory adaptation. After prolonged stimulation of these neurons, EGL-4 translocates from the cytosol to the nuclei of the AWC. This nuclear translocation event is both necessary and sufficient for adaptation of the AWC neuron to odor. A cGMP binding motif within EGL-4 and the Gα protein ODR-3 are both required for this translocation event, while loss of the guanylyl cyclase ODR-1 was shown to result in constitutively nuclear localization of EGL-4. However, the molecular changes that are integrated over time to produce a stably adapted response in the AWC are unknown. Here we show that odor-induced fluctuations in cGMP levels in the adult cilia may be responsible in part for sending EGL-4 into the AWC nucleus to produce long-term adaptation. We found that reductions in cGMP that result from mutations in the genes encoding the cilia-localized guanylyl cyclases ODR-1 and DAF-11 result in constitutively nuclear EGL-4 even in naive animals. Conversely, increases in cGMP levels that result from mutations in cGMP phosphodiesterases block EGL-4 nuclear entry even after prolonged odor exposure. Expression of a single phosphodiesterase in adult, naive animals was sufficient to modestly increase the number of animals with nuclear EGL-4. Further, coincident acute treatment of animals with odor and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) decreased the number of animals with nuclear EGL-4. These data suggest that reducing cGMP levels in AWC is necessary and even partially sufficient for nuclear translocation of EGL-4 and adaptation as a result of prolonged odor exposure. Our genetic analysis and chemical treatment of C. elegans further indicate that cilia morphology, as defined by fluorescent microscopic observation of the sensory endings, may allow for odor-induced fluctuations in cGMP levels and this fluctuation may be responsible for sending EGL-4 into the AWC nucleus. 相似文献
98.
99.
Bruder JT Semenova E Chen P Limbach K Patterson NB Stefaniak ME Konovalova S Thomas C Hamilton M King CR Richie TL Doolan DL 《PloS one》2012,7(4):e33920
The development of an effective malaria vaccine is a high global health priority. Vaccine vectors based on adenovirus type 5 are capable of generating robust and protective T cell and antibody responses in animal models and are currently being evaluated in clinical trials for HIV and malaria. They appear to be more effective in terms of inducing antigen-specific immune responses as compared with non-Ad5 serotype vectors. However, the high prevalence of neutralizing antibodies to Ad5 in the human population, particularly in the developing world, has the potential to limit the effectiveness of Ad5-based vaccines. We have generated novel Ad5-based vectors that precisely replace the hexon hypervariable regions with those derived from Ad43, a subgroup D serotype with low prevalence of neutralizing antibody in humans. We have demonstrated that these hexon-modified adenovectors are not neutralized efficiently by Ad5 neutralizing antibodies in vitro using sera from mice, rabbits and human volunteers. We have also generated hexon-modified adenovectors that express a rodent malaria parasite antigen, PyCSP, and demonstrated that they are as immunogenic as an unmodified vector. Furthermore, in contrast to the unmodified vector, the hexon-modified adenovectors induced robust T cell responses in mice with high levels of Ad5 neutralizing antibody. We also show that the hexon-modified vector can be combined with unmodified Ad5 vector in prime-boost regimens to induce protective responses in mice. Our data establish that these hexon-modified vectors are highly immunogenic even in the presence of pre-existing anti-adenovirus antibodies. These hexon-modified adenovectors may have advantages in sub-Saharan Africa where there is a high prevalence of Ad5 neutralizing antibody in the population. 相似文献
100.
Cutting edge: the dependence of plasma cells and independence of memory B cells on BAFF and APRIL 总被引:3,自引:0,他引:3
Benson MJ Dillon SR Castigli E Geha RS Xu S Lam KP Noelle RJ 《Journal of immunology (Baltimore, Md. : 1950)》2008,180(6):3655-3659
Memory B (B(MEM)) cells and long-lived bone marrow plasma cells (BM-PCs) persist within local environmental survival niches that afford cellular longevity. However, the factors supporting B(MEM) cell survival within the secondary lymphoid organs and allowing BM-PC persistence in the bone marrow remain poorly characterized. We report herein that long-lived B(MEM) cell survival and function are completely independent of BAFF (B cell-activating factor of the TNF family) or APRIL (a proliferation-inducing ligand). Thus, B(MEM) cells represent the only mature B2 lineage subset whose survival is independent of these ligands. We have previously shown that the TNFR family member receptor BCMA (B cell maturation Ag) is a critical survival receptor for BM-PC survival in vivo. We identify in this study the ligands critical for BM-PC survival and show that either BAFF or APRIL supports the survival of BM-PCs in vivo. These data define the BAFF/APRIL-dependent and -independent components of long-lived humoral immunity. 相似文献