GDP Dissociation Inhibitor Beta Research Articles

Differential protein expression in mouse splenic mononuclear cells treated with polysaccharides from spores of Ganoderma lucidum

Polysaccharides were one of the main components of Ganoderma lucidum, a medicinal mushroom well known for its immuno-modulation effects. In the present study, we demonstrated that polysaccharides extracted from Ganoderma lucidum spores (GL-SP) could stimulate splenic mononuclear cells (MNCs) proliferation and cytokine production. To identify the possible cellular targets of GL-SP in MNCs, two-dimensional gel electrophoresis (2-DE)-based comparative proteomics was performed and proteins altered in expressional level after GL-SP treatment were identified by MALDI-TOF MS/MS. Ten proteins with >2-fold increase or decrease expression in GL-SP-treated MNCs compared with control were found and further identified by MALDI-TOF MS/MS analysis and database searching. In the GL-SP-treated MNCs, there were increases in the expression of myosin regulatory light chain 2-A, Rho GDP dissociation inhibitor beta, T-cell-specific GTPase, phosphatidylinositol transfer protein alpha, and decreases in the expression of apoptosis-associated speck-like protein containing a CARD, 14-3-3 tau, beta-actin, tubulin alpha 2, copine I, and gamma-actin. The results of the present study help to provide insight into the immuno-modulating activities of GL-SP.

Differential Proteomics Analysis of Synaptic Proteins Identifies Potential Cellular Targets and Protein Mediators of Synaptic Neuroprotection Conferred by the Slow Wallerian Degeneration (Wlds) Gene

Non-somatic synaptic and axonal compartments of neurons are primary pathological targets in many neurodegenerative conditions, ranging from Alzheimer disease through to motor neuron disease. Axons and synapses are protected from degeneration by the slow Wallerian degeneration (Wld(s)) gene. Significantly the molecular mechanisms through which this spontaneous genetic mutation delays degeneration remain controversial, and the downstream protein targets of Wld(s) resident in non-somatic compartments remain unknown. In this study we used differential proteomics analysis to identify proteins whose expression levels were significantly altered in isolated synaptic preparations from the striatum of Wld(s) mice. Eight of the 16 proteins we identified as having modified expression levels in Wld(s) synapses are known regulators of mitochondrial stability and degeneration (including VDAC1, Aralar1, and mitofilin). Subsequent analyses demonstrated that other key mitochondrial proteins, not identified in our initial screen, are also modified in Wld(s) synapses. Of the non-mitochondrial proteins identified, several have been implicated in neurodegenerative diseases where synapses and axons are primary pathological targets (including DRP-2 and Rab GDP dissociation inhibitor beta). In addition, we show that downstream protein changes can be identified in pathways corresponding to both Ube4b (including UBE1) and Nmnat1 (including VDAC1 and Aralar1) components of the chimeric Wld(s) gene, suggesting that full-length Wld(s) protein is required to elicit maximal changes in synaptic proteins. We conclude that altered mitochondrial responses to degenerative stimuli are likely to play an important role in the neuroprotective Wld(s) phenotype and that targeting proteins identified in the current study may lead to novel therapies for the treatment of neurodegenerative diseases in humans.

Identification of nasopharyngeal carcinoma antigens that induce humoral immune response by proteomic analysis

A proteomics-based approach has been used to identify proteins that commonly elicit a humoral immune response in nasopharyngeal carcinoma (NPC). Sera from 19 newly diagnosed NPC patients and 19 healthy individuals were analyzed for IgG autoantibodies against NPC proteins resolved by 2-DE. Protein spots that exhibited selective reactivity with sera from NPC patients were identified by MS. Among nine identified proteins, cytokeratin 19 (CK19), Erb3 binding protein (EBP1), and Rho GDP dissociation inhibitor-beta (Rho-GDI-2) induced autoantibodies in more than 36.8% of NPC patients but not in healthy individuals. Furthermore, Western blot analysis and immunohistochemical staining were performed to determine the expression and localization of CK19, EBP1, and Rho-GDI-2 in NPC and normal nasopharyngeal mucosal tissues. Up-regulated CK19 and EBP1, but not Rho-GDI-2, were observed in NPC vs. normal tissue. Subcellular localization of the three proteins in NPC tissue was same as that in the normal tissue. Thus, overexpression of CK19 and EBP1 may be one of the mechanisms for their autoantibody development in NPC. To validate the findings of a proteomic analysis, occurrence of autoantibodies against these three proteins was detected by immunoprecipitation and Western blot analysis in additional 30 NPC patients, 23 other types of cancer patients and 20 healthy individuals. Results showed that frequency of autoantibodies against CK19, EBP1 and Rho-GDI-2 in NPC patients was significantly higher than that in other types of cancer patients and healthy individuals. We conclude that CK19, EBP1 and Rho-GDI-2 may have utility in NPC screening and diagnosis.

Comparative proteomic analysis of apoptosis induced by sodium selenite in human acute promyelocytic leukemia NB4 cells

Selenium (Se) is an essential trace element possessing anticarcinogenic properties. Sodium selenite (Na2SeO3) induced apoptosis in human acute promyelocytic leukemia (APL) cell line NB4 with dose and time dependency. In this study, proteomic techniques were used to study the apoptosis of NB4 cells induced by sodium selenite. Twenty-six downregulated and four upregulated proteins were identified, which exhibited a 1.5-fold change or greater. The identified proteins included key regulators of signal transduction such as Rho GDP dissociation inhibitor (Rho GDI) alpha and beta members of the MAPK family, and proteins involved in the regulation of c-fos or c-myc expression. Importantly, the identified proteins, hnRNP D0B and Rho GDI beta, which were related with the regulation of c-myc, c-fos, and c-jun, were determined by reverse transcription-polymerase chain reaction (RT-PCR) to confirm their downregulation in proteomic study. Western blot analysis and RT-PCR were then performed on three associated proteins: c-Myc, c-Fos, and c-Jun, and their expression were observed to be significantly downregulated. Results showed that certain regulation involved in c-myc, c-fos, and c-jun was present in the apoptosis, and the c-Myc dependent-on and Jun N-terminal kinase (JNK) pathway also play roles.

A Glutathione S-Transferase π-Activated Prodrug Causes Kinase Activation Concurrent with S-Glutathionylation of Proteins

Nitric oxide (NO) is an endogenous, diffusible, transcellular messenger shown to affect regulatory and signaling pathways with impact on cell survival. Exposure to NO can impart direct post-translational modifications on target proteins such as nitration and/or nitrosylation. As an alternative, after interaction with oxygen, superoxide, glutathione, or certain metals, NO can lead to S-glutathionylation, a post-translational modification potentially critical to signaling pathways. A novel glutathione S-transferase pi (GSTpi)-activated pro-drug, O(2)-[2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl]1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), liberates NO and elicits toxicity in vitro and in vivo. We now show that PABA/NO induces nitrosative stress, resulting in undetectable nitrosylation, limited nitration, and high levels of S-glutathionylation. After a single pharmacologically relevant dose of PABA/NO, S-glutathionylation occurs rapidly (<5 min) and is sustained for approximately 7 h, implying a half-life for the deglutathionylation process of approximately 3 h. Two-dimensional SDS-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody to S-glutathionylated residues indicated that numerous proteins were S-glutathionylated. Subsequent matrix-assisted laser desorption ionization/time of flight analysis identified 10 proteins, including beta-lactate dehydrogenase, Rho GDP dissociation inhibitor beta, ATP synthase, elongation factor 2, protein disulfide isomerase, nucleophosmin-1, chaperonin, actin, protein tyrosine phosphatase 1B (PTP1B), and glucosidase II. In addition, we showed that sustained S-glutathionylation was temporally concurrent with drug-induced activation of the stress kinases, known to be linked with cell death pathways. This is consistent with the fact that PABA/NO induces S-glutathionylation and inactivation of PTP1B, one phosphatase that can participate in deactivation of kinases. These effects were consistent with the presence of intracellular PABA/NO or metabolites, because cells overexpressing MRP1 were less sensitive to the drug and had reduced levels of S-glutathionylated proteins.

Holistic differential analysis of embryo‐induced alterations in the proteome of bovine endometrium in the preattachment period

During the peri-implantation period, molecular signaling between embryo and endometrium (layer of tissue lining the uterus lumen) is supposed to be crucial for the maintenance of pregnancy. To investigate embryo-induced alterations in the proteome of bovine endometrium in the preattachment period (day 18), we used monozygotic cattle twins (generated by embryo splitting) as a model eliminating genetic variability as a source for proteome differences. One of the twins was pregnant after the transfer of two in vitro produced blastocysts, while the corresponding twin received a sham-transfer and served as a nonpregnant control. The two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis of the endometrium samples of three twin pairs (pregnant/nonpregnant) revealed four proteins with significantly higher abundance (p < 10(-9)) in each sample derived from the pregnant animals: Rho GDP dissociation inhibitor beta; 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD); soluble NADP(+)-dependent isocitrate dehydrogenase 1; and acyl-CoA-binding protein. To verify the accuracy of the 2-D DIGE quantification, the abundances of 20 alpha-HSD were quantified by a targeted cleavable isotope-coded affinity tag (ICAT) approach. The mass spectrometry-based ICAT quantification matched perfectly the results obtained by 2-D DIGE quantification, demonstrating the accuracy of our data. These results demonstrate that our model (monozygotic twins) in combination with the appropriate analytical tools is particularly suitable for the detection of the proteins involved in the embryo-maternal interactions.

Overexpression of autocrine motility factor in metastatic tumor cells: possible association with augmented expression of KIF3A and GDI-β

Autocrine motility factor (AMF), which is identical to phosphohexose isomerase (PHI)/glucose-6-phosphate isomerase (GPI) , a ubiquitous enzyme essential for glycolysis, neuroleukin (NLK), a neurotrophic growth factor, and maturation factor (MF) mediating the differentiation of human myeloid cells, enhances the motility and metastatic ability of tumor cells. AMF/PHI activity is elevated in the serum or urine in patients with malignant tumors. Here, we constructed an amf/phi/nlk/mf gene using adenovirus vector and transfected into two tumor cell lines. Overexpression of AMF/PHI/NLK/MF enhanced AMF secretion into the culture media in both tumor cell lines. However, upregulation of motility and metastatic ability was found only in metastatic fibrosarcoma cells expressing an AMF receptor, gp78, and was not found in gp78-undetectable osteosarcoma cells. Thus, not only serum AMF activity but also gp78-expression in tumor cells may be required for metastasis-related motility induction. With the use of microarray analyses, we detected two augmented genes, rho GDP dissociation inhibitor beta and kinesin motor 3A, as well as AMF itself. The RNA message and protein expression of these two molecules was confirmed to be upregulated, suggesting a possible association with AMF-induced signaling for cell motility and metastasis.

LyGDI functions in cancer metastasis by anchoring Rho proteins to the cell membrane.

Rho family GTPases play an important role in a number of processes related to metastasis, and RhoGDP dissociation, inhibitors (RhoGDIs) regulate Rho family proteins. We cloned genomic DNA from colon carcinoma SW480 cells capable of transforming nonmetastatic ras-transformed 1-1ras1000 cells into metastatic cells. This DNA contained a truncated human ras homolog gene family GDP dissociation inhibitor beta (ARHGDIB) gene, resulting in a C-terminal truncated form of LyGDI (Delta C-LyGDI, 166-201 deletion), a member of the RhoGDIs. The stable expression of Delta C-LyGDI induced pulmonary metastasis in 1-1ras1000 cells, whereas expression of full-length LyGDI did not induce metastasis. Delta C-LyGDI was preferentially localized in the membrane, detected in a NP-40-insoluble fraction, and co-purified with radixin, moesin, Rac1, Cdc42, and RhoA. In Delta C-LyGDI transfectant, an activation state of Rac1 was elevated and Delta C-LyGDI was associated with Rac1-GTP. In keeping with the observed localization of Rac1 to the cell membrane and the elevated level of Rac1-GTP, Delta C-LyGDI transfectants were found to be more invasive than mock transfectant. These results suggest that LyGDI functions in the cell membrane to afford spatial regulation of Rho family GTPase signaling through ezrin radixin moesin (ERM) proteins during metastasis.

Proteomic identification of age-dependent protein nitration in rat skeletal muscle.

Age-related protein nitration was studied in skeletal muscle of Fisher 344 and Fisher 344/Brown Norway (BN) F1 rats by a proteomic approach. Proteins from young (4 months) and old (24 months) Fisher 344 rats and young (6 months) and old (34 months) Fisher 344/BN F1 animals were separated by 2-D gel electrophoresis. Western blot showed an age-related increase in the nitration of a few specific proteins, which were identified by MALDI-TOF MS and ESI-MS/MS. We identified age-dependent apparent nitration of beta-enolase, alpha-fructose aldolase, and creatine kinase, which perform important functions in muscle energy metabolism, suggesting that the nitration of such key proteins can be, in part, responsible for the decline of muscle motor function of the muscle. Furthermore, we have identified the apparent nitration of succinate dehydrogenase, rab GDP dissociation inhibitor beta (GdI-2), triosephosphate isomerase, troponin I, alpha-crystallin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Reduction of nucleoside diphosphate kinase B, Rab GDP-dissociation inhibitor beta and histidine triad nucleotide-binding protein in fetal Down syndrome brain.

Information on the various factors leading to impairments in the developing brain of fetal Down Syndrome patients is limited to few histological reports. We therefore attempted to describe expression levels of proteins in brain using the proteomic technique of two-dimensional electrophoresis with subsequent mass spectroscopical identification of protein spots and quantification with specific software. Cortical tissue was obtained from autopsy of human fetal abortus. Protein levels of GTP-binding nuclear protein ran, guanine nucleotide-binding protein g(o), alpha subunit 2, guanine nucleotide-binding protein g(i)/g(s)/g(t) beta subunit 1, -beta subunit 2, guanine nucleotide-binding protein beta subunit 5, nucleoside diphosphate kinase A, nucleoside diphosphate kinase B, Rab GDP-dissociation inhibitor beta, Rho GDP-dissociation inhibitor 1, biphosphate 3'-nucleotidase, small glutamine-rich tetra-tricopeptide repeat-containing protein and histidine triad nucleotide-binding protein were studied. Quantification revealed statistically significant reduced levels of nucleoside diphosphate kinase B, Rab GDP-dissociation inhibitor beta and histidine triad nucleotide-binding protein in fetal DS brain as compared to controls. We conclude that in early prenatal life proteins involved in neural differentiation, migration and synaptic transmission are impaired in DS cortex. These results may help to understand the abundant mechanisms leading to abnormalities in the wiring, structure and function of DS brain.

Two-hybrid System Screen with the Small GTP-binding Protein Rab6. IDENTIFICATION OF A NOVEL MOUSE GDP DISSOCIATION INHIBITOR ISOFORM AND TWO OTHER POTENTIAL PARTNERS OF Rab6

Rab6 is a small GTP-binding protein that belongs to the Ras superfamily and is involved in intra-Golgi transport. Using a two-hybrid system screen of a mouse brain cDNA library, we have isolated several clones encoding proteins that interact with Rab6. Approximately 60% of the clones identified encoded a new mouse Rab GDP dissociation inhibitor (GDI) isoform. This GDI isoform is distinct from mouse mGDI-1 and mGDI-2, which have been characterized previously, and most likely represents the mouse counterpart of the rat Rab GDI beta isoform. In the two-hybrid system, GDI beta interacts with wild-type Rab6 and Rab5, but not with a GTP-bound Rab6 mutant, or a Rab6 mutant that cannot be post-translationally processed. We further examined whether mouse GDI beta is functional; we show that recombinant mouse GDI beta is able to remove several Rab proteins, including Rab1, Rab2, Rab4, and Rab6, from membranes. The identification of a third GDI isoform in mouse raised the question whether GDI genes belong to a larger multigenic family. We have shown, by Southern blot analysis of genomic DNA, that at least five GDI gene copies exist in both the mouse and rat genomes. In our two-hybrid screen, we have also characterized another clone that specifically interacts with Rab6. This clone was partially sequenced but shows no homology to known sequences. Finally, a third clone, interacting with both Rab5 and Rab6, also appears to encode a novel protein.

Molecular cloning and characterization of two rab GDI species from rat brain: brain-specific and ubiquitous types.

Rab GDP dissociation inhibitor (GDI) is a regulatory protein for Rab proteins that controls not only the GDP/GTP exchange reaction of Rab proteins but also their translocation between the cytosol and cell membranes. Recently, rab GDI cDNAs have been isolated from human, bovine, rat, and Drosophila, and these Rab GDI proteins indicated an important role in the regulation of intracellular vesicle traffic. In this study, we have isolated two different rab GDI cDNAs, designated rat rab GDI alpha and beta, from a rat brain cDNA library using bovine rab GDI cDNA as a probe. Rat rab GDI alpha and beta show 99 and 86% amino acid identity with bovine rab GDI, respectively, indicating that rat rab GDI alpha is a rat counterpart of bovine rab GDI. Both rat Rab GDI alpha and beta proteins expressed in Escherichia coli showed a similar degree of activity of regulating the GDP/GTP exchange reaction to that of bovine Rab GDI using Rab3A and Rab11 as substrates. Northern blot analysis revealed that rab GDI alpha mRNA was expressed abundantly in brain but much less in other tissues, whereas rat rab GDI beta mRNA was ubiquitously expressed. Reverse transcription-polymerase chain reaction analysis revealed that astrocytes expressed rat rab GDI beta gene but not rat rab GDI alpha gene. These results indicate that Rab GDI alpha is involved in sorting of highly specialized vesicles in brain such as neurosecretory vesicles, whereas Rab GDI beta plays a general role in vesicular trafficking in diverse types of cells.