Proteomic analysis of astrocytic secretion in the mouse. Comparison with the cerebrospinal fluid proteome

Astrocytes, the most abundant cell type in the central nervous system, are intimately associated with synapses. They play a pivotal role in neuronal survival and the brain inflammatory response. Some astrocytic functions are mediated by the secretion of polypeptides. Using a proteomic approach, we have identified more than 30 proteins released by cultured astrocytes. These include proteases and protease inhibitors, carrier proteins, and antioxidant proteins. Exposing astrocytes to brefeldin A, which selectively blocks secretory vesicle assembly, suppressed the release of some of these proteins. This indicates that astrocytes secrete these proteins by a classic vesicular mechanism and others by an alternative pathway. Astrocytes isolated from different brain regions secreted a similar pattern of proteins. However, the secretion of some of them, including metalloproteinase inhibitors and apolipoprotein E, was region-specific. In addition, pro-inflammatory treatments modified the profile of astrocytic protein secretion. Finally, more than two thirds of the proteins identified in the astrocyte-conditioned medium were detectable in the mouse cerebrospinal fluid, suggesting that astrocytes contribute to the cerebrospinal fluid protein content. In conclusion, this study provides the first unbiased characterization of the major proteins released by astrocytes, which may play a crucial role in the modulation of neuronal survival and function.     

Peroxidation of liposomal palmitoyllinoleoylphosphatidylcholine (PLPC), effects of surface charge on the oxidizability and on the potency of antioxidants

Peroxidation of membrane phospholipids is an important determinant of membrane function. Previously we studied the kinetics of peroxidation of the polyunsaturated fatty acid (PUFA) residues in model membranes (liposomes) made by sonication of palmitoyllinoleoylphosphatidylcholine (PLPC). Since most biomembranes are negatively-charged, we have now studied the effect of negative surface charge on the kinetics of peroxidation of liposomes made of PLPC and 9% of one of the negatively-charged phospholipids phosphatidylserine (PS) or phosphatidic acid (PA). Peroxidation was initiated by either CuCl2 or AAPH and continuously monitored spectrophotometrically. The following results were obtained: (i) The negative charge had only a slight effect on AAPH-induced peroxidation, but accelerated markedly copper-induced peroxidation of the liposomes, probably by increasing the binding of copper to the membrane surface. (ii) Ascorbic acid (AA) inhibited AAPH-induced but promoted copper-induced peroxidation in all the studied liposomes, probably by enhancing the production of free radicals upon reduction of Cu(II) to Cu(I). (iii) alpha-tocopherol (Toc) inhibited AAPH-induced peroxidation in all the studied liposomes, whereas the effect of tocopherol on copper-induced peroxidation varied from being pro-oxidative in PA-containing liposomes, to being extremely anti-oxidative in PS-containing liposomes, even at very low tocopherol concentrations. The significance of the latter unusual protective effect, which we attribute to recycling of tocopherol by a PS-Cu complex, requires further investigation.     

Synthesis and localization of the Salmonella SPI-1 type III secretion needle complex proteins PrgI and PrgJ

An essential component of type III secretion systems (TTSS) is a supramolecular structure termed the needle complex. In Salmonella enterica, at least four proteins make up this structure: InvG, PrgH, PrgK, and PrgI. Another protein, PrgJ, is thought to play a role in the assembly of this structure, but its function is poorly understood. We have analyzed the expression and localization of PrgJ and the needle protein PrgI in different S. enterica serovar Typhimurium mutant strains. We found that the levels of PrgI and PrgJ were significantly reduced in a TTSS-deficient invA mutant strain and that the decreased levels were due to protein instability. In addition, we found that PrgJ, although associated with the needle complex in wild-type S. enterica serovar Typhimurium, was absent from needle complexes obtained from an invJ mutant strain, which exhibits very long needle substructures. We suggest that PrgJ is involved in capping the needle substructure of the needle complex.     

The dose-dependent effect of copper-chelating agents on the kinetics of peroxidation of low-density lipoprotein (LDL)

Copper-induced peroxidation of lipoproteins involves continuous production of free radicals via a redox cycle of copper. Formation of Cu(I) during Cu(II)-induced peroxidation of LDL was previously demonstrated by accumulation of the colored complexes of Cu(I) in the presence of one of the Cu(I)-specific chelators bathocuproine (BC) or neocuproine (NC). All the studies conducted thus far employed high concentrations of these chelators (chelator/Cu(II) > 10). Under these conditions, at low copper concentrations the chelators prolonged the lag preceding oxidation, whereas at high copper concentrations the chelators shortened the lag. In an attempt to gain understanding of these non-monotonic effects, we have studied systematically the peroxidation of LDL (0.1 microM, 50 microg protein/mL) at varying concentrations of NC or BC over a wide range of concentrations of the chelators and copper. These studies revealed that: (i) At copper concentrations of 5 microM and below, NC prolonged the lag in a monotonic, dose-dependent fashion typical for other complexing agents. However, unlike with other chelators, the maximal rate of oxidation was only slightly reduced (if at all). (ii) At copper concentrations of 15 microM and above, the addition of about 20 microM NC or BC resulted in prolongation of the lag, but this effect became smaller at higher concentrations of the chelators, and at yet higher concentrations the lag became much shorter than that observed in the absence of chelators. Throughout the whole range of NC concentrations, the maximal rate of peroxidation increased monotonically upon increasing the NC concentration. (iii) Unlike in the absence of chelators, the prooxidative effect of copper did not exhibit saturation with respect to copper, up to copper concentrations of 30 microM. Based on these results we conclude that the copper-chelates can partition into the hydrophobic core of LDL particles and induce peroxidation by forming free radicals within the core. This may be significant with respect to the understanding of the possible mechanisms of peroxidation by chelated transition metals in vivo.     

Flow and image cytometry in thymic neoplasia: correlation with clinical outcome

OBJECTIVE: To compare nuclear DNA by flow (FCM) and image cytometry (ICM) in thymic neoplasms and to relate results to clinical outcome. STUDY DESIGN: DNA ploidy of 44 thymomas and 6 thymic carcinomas was studied by FCM and ICM of single nuclear suspensions from paraffin blocks. RESULTS: By FCM, 33 thymomas (75%) and one thymic carcinoma (17%) were diploid; 6 thymomas (14%) and 4 thymic carcinomas (67%) were aneuploid. By ICM, 36 thymomas (82%) were diploid; 7 thymomas (16%) and 6 thymic carcinomas (100%) were aneuploid. Mean follow-up in 44 cases was 46.2 months (range, 1-162). Ten patients with persistent/recurrent disease included four with thymic carcinoma, who died of the disease (two aneuploid by both techniques, two aneuploid by ICM with unsatisfactory/diploid FCM). Four had invasive thymoma and recurrence after 13-150 months (two diploid and two aneuploid by both methods), one had diploidy and noninvasive thymoma that recurred at 92 months, and one had an epithelial thymoma that recurred at 144 months (aneuploid by FCM, diploid by ICM). CONCLUSION: The results obtained in this preliminary, retrospective study show a high concordance between FCM and ICM; aneuploidy correlated with poor outcome by both methodologies. While these findings are encouraging, larger numbers of cases will be needed to define the role of FCM and ICM in predicting outcome in thymic tumors.     

Copper-induced peroxidation of liposomal palmitoyllinoleoylphosphatidylcholine (PLPC), effect of antioxidants and its dependence on the oxidative stress

In an attempt to deepen our understanding of the mechanisms responsible for lipoprotein peroxidation, we have studied the kinetics of copper-induced peroxidation of the polyunsaturated fatty acid residues in model membranes (small, unilamellar liposomes) composed of palmitoyllinoleoylphosphatidylcholine (PLPC). Liposomes were prepared by sonication and exposed to CuCl(2) in the absence or presence of naturally occurring reductants (ascorbic acid (AA) and/or alpha-tocopherol (Toc)) and/or a Cu(I) chelator (bathocuproinedisulfonic acid (BC) or neocuproine (NC)). The resultant oxidation process was monitored by recording the time-dependence of the absorbance at several wavelengths. The observed results reveal that copper-induced peroxidation of PLPC is very slow even at relatively high copper concentrations, but occurs rapidly in the presence of ascorbate, even at sub-micromolar copper concentrations. When added from an ethanolic solution, tocopherol had similar pro-oxidative effects, whereas when introduced into the liposomes by co-sonication tocopherol exhibited a marked antioxidative effect. Under the latter conditions, ascorbate inhibited peroxidation of the tocopherol-containing bilayers possibly by regeneration of tocopherol. Similarly, both ascorbate and tocopherol exhibit antioxidative potency when the PLPC liposomes are exposed to the high oxidative stress imposed by chelated copper, which is more redox-active than free copper. The biological significance of these results has yet to be evaluated.     

Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE

The bacterial enteropathogen Salmonella typhimurium employs a type III secretion system to inject bacterial toxins into the host cell cytosol. These toxins transiently activate Rho family GTP-binding protein-dependent signaling cascades to induce cytoskeletal rearrangements. One of these translocated Salmonella toxins, SopE, can activate Cdc42 in a Dbl-like fashion despite its lack of sequence similarity to Dbl-like proteins, the Rho-specific eukaryotic guanine nucleotide exchange factors. To elucidate the mechanism of SopE-mediated guanine nucleotide exchange, we have analyzed the structure of the complex between a catalytic fragment of SopE and Cdc42. SopE binds to and locks the switch I and switch II regions of Cdc42 in a conformation that promotes guanine nucleotide release. This conformation is strikingly similar to that of Rac1 in complex with the eukaryotic Dbl-like exchange factor Tiam1. However, the catalytic domain of SopE has an entirely different architecture from that of Tiam1 and interacts with the switch regions via different amino acids. Therefore, SopE represents the first example of a non-Dbl-like protein capable of inducing guanine nucleotide exchange in Rho family proteins.     

A proteomic approach based on peptide affinity chromatography, 2-dimensional electrophoresis and mass spectrometry to identify multiprotein complexes interacting with membrane-bound receptors

There is accumulating evidence that membrane-bound receptors interact with many intracellular proteins. Multiprotein complexes associated with ionotropic receptors have been extensively characterized, but the identification of proteins interacting with G protein-coupled receptors (GPCRs) has so far only been achieved in a piecemeal fashion, focusing on one or two protein species. We describe a method based on peptide affinity chromatography, two-dimensional electrophoresis, mass spectrometry and immunoblotting to identify the components of multiprotein complexes interacting directly or indirectly with intracellular domains of GPCRs or, more generally, any other membrane-bound receptor. Using this global approach, we have characterized multiprotein complexes that bind to the carboxy-terminal tail of the 5-hydroxytryptamine type 2C receptor and are important for its subcellular localization in CNS cells (Bécamel et al., EMBO J., 21(10): 2332, 2002). Published: December 9, 2002     

Salt bridges at the inter-ring interface regulate the thermostat of GroEL

The chaperonin GroEL consists of a double-ring structure made of identical subunits and displays unusual allosteric properties caused by the interaction between its constituent subunits. Cooperative binding of ATP to a protein ring allows binding of GroES to that ring, and at the same time negative inter-ring cooperativity discharges the ligands from the opposite ring, thus driving the protein-folding cycle. Biochemical and electron microscopy analysis of wild type GroEL, a single-ring mutant (SR1), and two mutants with one inter-ring salt bridge of the chaperonin disrupted (E461K and E434K) indicate that these ion pairs form part of the interactions that allow the inter-ring allosteric signal to be transmitted. The wild type-like activities of the ion pair mutants at 25 degrees C are in contrast with their lack of inter-ring communication and folding activity at physiological temperatures. These salt bridges stabilize the inter-ring interface and maintain the inter-ring spacing so that functional communication between protein heptamers takes place. The characterization of GroEL hybrids containing different amounts of wild type and mutant subunits also indicates that as the number of inter-ring salt bridges increases the functional properties of the hybrids recover. Taken together, these results strongly suggest that inter-ring salt bridges form a stabilizing ring-shaped, ionic zipper that ensures inter-ring communication at the contact sites and therefore a functional protein-folding cycle. Furthermore, they regulate the chaperonin thermostat, allowing GroEL to distinguish physiological (37 degrees C) from stress temperatures (42 degrees C).