Rab GDP Dissociation Inhibitor Beta Research Articles

Modulation of Rab GDP-Dissociation Inhibitor Trafficking and Expression by the Transmembrane Protein 59 (TMEM59)

Transmembrane protein 59 (TMEM59) is a type I transmembrane protein. However, the characterization and functions of TMEM59 in cells are not clear. Our results showed that TMEM59 localizes to vesicular structures. Further co-localization studies illustrated that TMEM59 is mainly distributed in the lysosome and acidic vesicular. TMEM59 movement between the nucleus and cell membrane was observed in living cells expressing TMEM59–EGFP fusion proteins. In addition, cell surface transport of amyloid precursor protein (APP) was significantly inhibited by TMEM59 and increased APP levels in HEK296T cells. TMEM59 also significantly inhibits transport of Rab GDP dissociation inhibitor alpha (GDI1) and Rab GDP dissociation inhibitor beta (GDI2), and further increases expression of GDI1 and GDI2 proteins in the cytoplasm. However, TMEM59 does not affect protein expression and localization of BACE2. These results suggest that TMEM59 may be involved in the packaging of acidic vesicles, modulated transport, and processing of APP, GDI1, and GDI2.

The characterization of transmembrane protein 59-like (TMEM59L) reveals its role in the regulating the level of the GDI protein family

The characterization and functions of transmembrane protein 59-like (TMEM59L), a type I transmembrane protein, are not clearly understood until now. Some TMEM59L and fluorescent fusion proteins constructs were transfected in cell lines and liposomes, and their localization was observed. The effects of protein constructs were studied by fluorescence microscopy and western blotting. This study reports a novel function of human TMEM59L (hTMEM59L) related to the expression and location of some proteins. In addition, we report two novel splice variants of human TMEM59L (hTMEM59L). The localization of mutants of this protein, lacking a middle region, and a C-terminal deletion, markedly differed from that of full-length hTMEM59L. Intracellular movement assessment in living cells showed the localization of TMEM59L to vesicular structures in the Golgi bodies, and the cell membrane was observed in living cells. Overexpression of TMEM59L markedly increased the level of amyloid precursor protein because of TMEM59L-mediated inhibition of cell membrane transport but did not affect the expression of beta-secretase 2. TMEM59L overexpression also significantly increased the levels of Rab GDP dissociation inhibitor alpha and Rab GDP dissociation inhibitor beta proteins. These results suggest that TMEM59L is involved in the packaging of acidic vesicles and thereby functions in the trafficking and processing of intracellular proteins.

ISGylation drives basal breast tumour progression by promoting EGFR recycling and Akt signalling

ISG15 is an ubiquitin-like modifier that is associated with reduced survival rates in breast cancer patients. The mechanism by which ISG15 achieves this however remains elusive. We demonstrate that modification of Rab GDP-Dissociation Inhibitor Beta (GDI2) by ISG15 (ISGylation) alters endocytic recycling of the EGF receptor (EGFR) in non-interferon stimulated cells using CRISPR-knock out models for ISGylation. By regulating EGFR trafficking, ISGylation enhances EGFR recycling and sustains Akt-signalling. We further show that Akt signalling positively correlates with levels of ISG15 and its E2-ligase in basal breast cancer cohorts, confirming the link between ISGylation and Akt signalling in human tumours. Persistent and enhanced Akt activation explains the more aggressive tumour behaviour observed in human breast cancers. We show that ISGylation can act as a driver of tumour progression rather than merely being a bystander.

Proteomic and phosphoproteomic analyses reveal several events involved in the early stages of bovine herpesvirus 1 infection.

Herpesviruses are predicted to express more than 80 proteins during their infection cycle. The proteins synthesized by the immediate early genes and early genes target signaling pathways in host cells that are essential for the successful initiation of a productive infection and for latency. In this study, proteomic and phosphoproteomic tools showed the occurrence of changes inMadin-Darby bovine kidney cells at the early stageof the infectionbybovine herpesvirus 1 (BoHV-1). Proteins that had already been described in the early stage of infection for other herpesviruses but not for BoHV-1 were found. For example, stathmin phosphorylation at the initial stage of infection is described for the first time. In addition, two proteins that had not been described yet in the early stages of herpesvirus infections in general were ribonuclease/angiogenin inhibitor and Rab GDP dissociation inhibitor beta. The biological processes involved in these cellular responses were repair and replication of DNA, splicing, microtubule dynamics, and inflammatory responses. These results reveal pathways that might be used as targets for designing antiviral molecules against BoHV-1 infection.

Tetracapsuloides bryosalmonae infection affects the expression of genes involved in cellular signal transduction and iron metabolism in the kidney of the brown trout Salmo trutta.

Tetracapsuloides bryosalmonae is an enigmatic endoparasite which causes proliferative kidney disease in various species of salmonids in Europe and North America. The life cycle of the European strain of T. bryosalmonae generally completes in an invertebrate host freshwater bryozoan and vertebrate host brown trout (Salmo trutta) Linnaeus, 1758. Little is known about the gene expression in the kidney of brown trout during the developmental stages of T. bryosalmonae. In the present study, quantitative real-time PCR was applied to quantify the target genes of interest in the kidney of brown trout at different time points of T. bryosalmonae development. PCR primers specific for target genes were designed and optimized, and their gene expression levels were quantified in the cDNA kidney samples using SYBR Green Supermix. Expression of Rab GDP dissociation inhibitor beta, integral membrane protein 2B, NADH dehydrogenase 1 beta subcomplex subunit 6, and 26S protease regulatory subunit S10B were upregulated significantly in infected brown trout, while the expression of the ferritin M middle subunit was downregulated significantly. These results suggest that host genes involved in cellular signal transduction, proteasomal activities, including membrane transporters and cellular iron storage, are differentially upregulated or downregulated in the kidney of brown trout during parasite development. The gene expression pattern of infected renal tissue may support the development of intraluminal sporogonic stages of T. bryosalmonae in the renal tubular lumen of brown trout which may facilitate the release of viable parasite spores to transmit to the invertebrate host bryozoan.

Usefulness of DIGE for the detection of protein profile in retained and released bovine placental tissues

IntroductionRegardless intensive research, the etiology and mechanisms of retention of fetal membranes in cows, still require elucidation. In our research approach, difference in gel electrophoresis (DIGE) and matrix-assisted laser desorption/ionization (MALDI) identification were used to obtain first results on protein profile of bovine placental membranes which were properly released or retained for more than 12 h after parturition. MethodsPlacentomes from 6 cows that released placenta and from 6 cows that retained fetal membranes were homogenized, fluorescence labeled and subjected to DIGE. ResultsSelected spots that significantly differed between retained and released placenta as well as spots with constant appearance were identified by MALDI. This allowed identification of the following proteins with high statistical reliability: Transforming growth factor beta 2 – high expression in maternal and fetal part of retained fetal membranes, Short transient receptor potential channel 5 –high expression in maternal part of retained and not retained fetal membranes, Rab GDP dissociation inhibitor beta – high expression in fetal part of retained and not retained fetal membranes, Proline dehydrogenase 2 – similar expression in all examined samples, Ras-related protein Rab-7b –high expression only in maternal part of not retained fetal membranes. DiscussionUp to now, these proteins have not been considered as possibly important molecules for the separation/retention of fetal membranes, but their biological roles may suggest it. Further studies are necessary to establish a full profile of bovine placental proteins and define target molecules that may be involved in separation/retention of fetal membranes.

Bioinformatics analysis of Rab GDP dissociation inhibitor beta and its expression in non-small cell lung cancer.

BackgroundLung cancer has been considered as one of the most important causes of cancer-related mortality worldwide. To predict lung cancer, researchers identified several molecular markers. However, many underlying markers of lung cancer remain unclear. One of these markers is Rab GDP dissociation inhibitor beta (GDIβ), which is related to tumorigenicity, development and invasion. This study was designed to analyze the biological characteristics of Rab GDIβ and to detect the mRNA and protein expressions of Rab GDIβ in lung cancer cells; this study also aimed to investigate the functions of this protein in lung cancer.MethodUsing online software from the websites of NCBI, ProtParam and so on, we analyzed the biological characteristics of Rab GDIβ. RT-PCR was performed to detect gene expressions in A549 and 16HBE cell lines and immunohistochemistry (IHC) staining was conducted to detect Rab GDIβ protein expression in 57 cases of human lung cancer tissues and 19 cases of normal lung tissues. The association of protein expression with patient clinical and pathological characteristics was assessed in each dataset.ResultsBioinformatic analysis on Rab GDIβ: The mRNA of human Rab GDIβ contains two transcript variants; the common structural elements of the two proteins are mainly α-helix, random coil, β-turn and extended strand. Three and four transmembrane domains could be found in the entire polypeptide chain of protein variants 1 and 2, respectively; both transcript variants are hydrophilic and soluble proteins. The RT-PCR result: The mRNA expression of Rab GDIβ was down-regulation in A549 cells compared with that in 16HBE cells. The IHC result: The protein expression of Rab GDIβ in lung cancer cells was significantly lower than that in normal lung tissues (P <0.05) but was not correlated with patients’ age, gender, tumor size, pathological type, differentiation, lymph node metastasis, distant metastasis and TNM stage.ConclusionThe expression of Rab GDIβ was low in non-small cell lung cancer (NSCLC). Hence, Rab GDIβ may be a tumor suppressor and could function as an indicator of tumorigenesis in NSCLC; nevertheless, this result should be further studied.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_201

Serological Proteome Analysis (SERPA) as a tool for the identification of new candidate autoantigens in type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease characterized by the presence of circulating autoantibodies directed against proteins of islet beta-cell. Autoantibody testing is used for diagnostic purposes; however, up to 2-5% of patients who are clinically diagnosed with T1D are found negative for known antibodies, suggesting that the T1D autoantigen panel is incomplete. With the aim of identifying new T1D autoantigen(s), we used sera from subjects clinically diagnosed with T1D, but who tested negative for the four T1D autoantibodies currently used in clinical practice and for genes responsible for sporadic cases of diabetes. Sera from these patients were challenged by Western blot against the proteome from human pancreatic beta-cells resolved by 2DE. Eleven proteins were identified by MS. A radiobinding assay (RBA) was developed to test the reactivity to Rab GDP dissociation inhibitor beta (GDIβ) of T1D sera using an independent method. Depending on the construct used (open reading frame or COOH-terminus) 22% to 32% of fifty T1D sera showed increased binding to GDIβ by RBA. In addition, 15% of patients with celiac disease had raised binding to the COOH-terminus GDIβ. These results indicate that immunoproteomics is a feasible strategy for the identification of candidate T1D autoantigens. Several approaches have been previously used to look for new type 1 diabetes autoantigens. With the present work we show that carefully selected sera from rare patients with diabetes both negative for the 5 autoantibodies currently used in clinical practice and for genes responsible for sporadic cases of diabetes, may be exploited in experiments utilizing human pancreatic islets extracts as a target for SERPA to identify novel candidate T1D autoantigens.

Tamarindus indica Extract Alters Release of Alpha Enolase, Apolipoprotein A-I, Transthyretin and Rab GDP Dissociation Inhibitor Beta from HepG2 Cells

BackgroundThe plasma cholesterol and triacylglycerol lowering effects of Tamarindus indica extract have been previously described. We have also shown that the methanol extract of T. indica fruit pulp altered the expression of lipid-associated genes including ABCG5 and APOAI in HepG2 cells. In the present study, effects of the same extract on the release of proteins from the cells were investigated using the proteomics approach.Methodology/Principal FindingsWhen culture media of HepG2 cells grown in the absence and presence of the methanol extract of T. indica fruit pulp were subjected to 2-dimensional gel electrophoresis, the expression of seven proteins was found to be significantly different (p<0.03125). Five of the spots were subsequently identified as alpha enolase (ENO1), transthyretin (TTR), apolipoprotein A-I (ApoA-I; two isoforms), and rab GDP dissociation inhibitor beta (GDI-2). A functional network of lipid metabolism, molecular transport and small molecule biochemistry that interconnects the three latter proteins with the interactomes was identified using the Ingenuity Pathways Analysis software.Conclusion/SignificanceThe methanol extract of T. indica fruit pulp altered the release of ENO1, ApoA-I, TTR and GDI-2 from HepG2 cells. Our results provide support on the effect of T. indica extract on cellular lipid metabolism, particularly that of cholesterol.

Sample complexity reduction aids efficient detection of low‐abundant proteins from human amniotic fluid

Working with biological fluids poses a challenge of visualizing proteins present in lower concentrations. This study describes a batch-mode chromatographic method for the fractionation of human amniotic fluid (AF). This method is easy to use with minimal sample quantity, resin volume and sample processing time. For albumin depletion, two methods were evaluated. The results demonstrated that specific depletion of albumin, using affinity-ligand-based resin, is more efficient than the conventional dye-based method. The albumin-depleted human AF was fractionated by strong anion-exchange resin in spin devices, for sample, complexity reduction and enrichment of low-abundant proteins. Analysis of four eluate fractions generated after this step shows enrichment of few low-abundant proteins. Two novel low-abundant proteins, Rab GDP dissociation inhibitor beta and peptide methionine sulfoxide reductase, were identified from human AF. Alpha-1-B glycoprotein was successfully identified by this strategy, whereas the published literature reports that it was not identified by strong anion-exchange FPLC followed by SDS-PAGE. Therefore, the current method has distinct advantages over the conventional column-based chromatography. This study also reports altered expression of some proteins in Rh-isoimmunized AF samples in comparison with normal AF.

Identification of metastasis-associated proteins involved in gallbladder carcinoma metastasis by proteomic analysis and functional exploration of chloride intracellular channel 1

Advanced gallbladder cancer has an extremely poor prognosis because of metastasis. Identification of metastasis-related biomarkers is essential to improve patient survival. In the present study, metastasis-associated proteins were identified by comparative proteomic analysis and the metastasis-related function of the candidate protein, chloride intracellular channel 1 (CLIC1), was further elucidated. Two cell lines with high or low metastatic potential (termed GBC-SD18H and GBC-SD18L, respectively), originating from the same parental gallbladder carcinoma GBC-SD cell line, were identified by spontaneous metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach comprised of two-dimensional gel electrophoresis analysis and mass spectroscopy was used to identify and compare the protein expression patterns between GBC-SD18L and GBC-SD18H. Twenty-six proteins were identified and further verified by one-dimensional Western blotting and semiquantitative reverse transcriptase polymerase chain reaction analysis. It was determined that CLIC1, ezrin, vimentin, annexin A3, WD repeat domain 1, triosephosphate isomerase, C1-tetrahydrofolate synthase, Rho GDP-dissociation inhibitor 1, T-complex protein 1, heterogeneous nuclear ribonucleoprotein K, glutamate dehydrogenase 1, proteasome activator complex subunit 3 and Rab GDP-dissociation inhibitor beta were significantly up-regulated in the highly metastatic GBC-SD18H cell line compared to the poorly metastatic GBC-SD18L cell line. However, phosphoglycerate kinase 1 and programmed cell death protein 8 were significantly down-regulated in the highly metastatic GBC-SD18H cell line compared to GBC-SD18L. Considering that CLIC1 was profuse in highly metastatic GBC-SD18H but scarce in poorly metastatic GBC-SD18L, the association of CLIC1 with metastasis was further elucidated by the overexpression and RNA interference of CLIC1 in GBC-SD18L cells and GBC-SD18H cells, respectively. The results demonstrated that the overexpression of CLIC1 promoted cell motility and invasion of GBC-SD18L in vitro, while RNA interference of CLIC1 remarkably decreased cell motility and invasive potency of GBC-SD18H in vitro, indicating that CLIC1 might play an important role in metastasis of gallbladder carcinoma.

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.

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.