Protein S-Bacillithiolation Functions in Thiol Protection and Redox Regulation of the Glyceraldehyde-3-Phosphate Dehydrogenase Gap in Staphylococcus aureus Under Hypochlorite Stress

PubMed Central

Imber, Marcel; Huyen, Nguyen Thi Thu; Pietrzyk-Brzezinska, Agnieszka J.; Loi, Vu Van; Hillion, Melanie; Bernhardt, Jörg; Thärichen, Lena; Kolšek, Katra; Saleh, Malek; Hamilton, Chris J.; Adrian, Lorenz; Gräter, Frauke; Wahl, Markus C.

2018-01-01

Abstract Aims: Bacillithiol (BSH) is the major low-molecular-weight thiol of the human pathogen Staphylococcus aureus. In this study, we used OxICAT and Voronoi redox treemaps to quantify hypochlorite-sensitive protein thiols in S. aureus USA300 and analyzed the role of BSH in protein S-bacillithiolation. Results: The OxICAT analyses enabled the quantification of 228 Cys residues in the redox proteome of S. aureus USA300. Hypochlorite stress resulted in >10% increased oxidation of 58 Cys residues (25.4%) in the thiol redox proteome. Among the highly oxidized sodium hypochlorite (NaOCl)-sensitive proteins are five S-bacillithiolated proteins (Gap, AldA, GuaB, RpmJ, and PpaC). The glyceraldehyde-3-phosphate (G3P) dehydrogenase Gap represents the most abundant S-bacillithiolated protein contributing 4% to the total Cys proteome. The active site Cys151 of Gap was very sensitive to overoxidation and irreversible inactivation by hydrogen peroxide (H2O2) or NaOCl in vitro. Treatment with H2O2 or NaOCl in the presence of BSH resulted in reversible Gap inactivation due to S-bacillithiolation, which could be regenerated by the bacilliredoxin Brx (SAUSA300_1321) in vitro. Molecular docking was used to model the S-bacillithiolated Gap active site, suggesting that formation of the BSH mixed disulfide does not require major structural changes. Conclusion and Innovation: Using OxICAT analyses, we identified 58 novel NaOCl-sensitive proteins in the pathogen S. aureus that could play protective roles against the host immune defense and include the glycolytic Gap as major target for S-bacillithiolation. S-bacillithiolation of Gap did not require structural changes, but efficiently functions in redox regulation and protection of the active site against irreversible overoxidation in S. aureus. Antioxid. Redox Signal. 28, 410–430. PMID:27967218

Protein S-Bacillithiolation Functions in Thiol Protection and Redox Regulation of the Glyceraldehyde-3-Phosphate Dehydrogenase Gap in Staphylococcus aureus Under Hypochlorite Stress.

PubMed

Imber, Marcel; Huyen, Nguyen Thi Thu; Pietrzyk-Brzezinska, Agnieszka J; Loi, Vu Van; Hillion, Melanie; Bernhardt, Jörg; Thärichen, Lena; Kolšek, Katra; Saleh, Malek; Hamilton, Chris J; Adrian, Lorenz; Gräter, Frauke; Wahl, Markus C; Antelmann, Haike

2018-02-20

Bacillithiol (BSH) is the major low-molecular-weight thiol of the human pathogen Staphylococcus aureus. In this study, we used OxICAT and Voronoi redox treemaps to quantify hypochlorite-sensitive protein thiols in S. aureus USA300 and analyzed the role of BSH in protein S-bacillithiolation. The OxICAT analyses enabled the quantification of 228 Cys residues in the redox proteome of S. aureus USA300. Hypochlorite stress resulted in >10% increased oxidation of 58 Cys residues (25.4%) in the thiol redox proteome. Among the highly oxidized sodium hypochlorite (NaOCl)-sensitive proteins are five S-bacillithiolated proteins (Gap, AldA, GuaB, RpmJ, and PpaC). The glyceraldehyde-3-phosphate (G3P) dehydrogenase Gap represents the most abundant S-bacillithiolated protein contributing 4% to the total Cys proteome. The active site Cys151 of Gap was very sensitive to overoxidation and irreversible inactivation by hydrogen peroxide (H 2 O 2 ) or NaOCl in vitro. Treatment with H 2 O 2 or NaOCl in the presence of BSH resulted in reversible Gap inactivation due to S-bacillithiolation, which could be regenerated by the bacilliredoxin Brx (SAUSA300_1321) in vitro. Molecular docking was used to model the S-bacillithiolated Gap active site, suggesting that formation of the BSH mixed disulfide does not require major structural changes. Conclusion and Innovation: Using OxICAT analyses, we identified 58 novel NaOCl-sensitive proteins in the pathogen S. aureus that could play protective roles against the host immune defense and include the glycolytic Gap as major target for S-bacillithiolation. S-bacillithiolation of Gap did not require structural changes, but efficiently functions in redox regulation and protection of the active site against irreversible overoxidation in S. aureus. Antioxid. Redox Signal. 28, 410-430.

Molecular Characterization of the SUMO-1 Modification of RanGAP1 and Its Role in Nuclear Envelope Association

PubMed Central

Mahajan, Rohit; Gerace, Larry; Melchior, Frauke

1998-01-01

The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping, mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101– amino acid protein are proteolytically removed before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo. In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE), where it was previously shown to be required for nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1–SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role. PMID:9442102

Homotypic gap junctional communication associated with metastasis increases suppression increases with PKA kinase activity and is unaffected by P13K inhibition

USDA-ARS?s Scientific Manuscript database

Loss of gap junctional intercellular communication (GJIC) between cancer cells is a common characteristic of malignant transformation. This communication is mediated by connexin proteins that make up the functional units of gap junctions. Connexins are highly regulated at the protein level and phosp...

Putative terminator and/or effector functions of Arf GAPs in the trafficking of clathrin-coated vesicles.

PubMed

Kon, Shunsuke; Funaki, Tomo; Satake, Masanobu

2011-05-01

The role of ArfGAP1 as a terminator or effector in COPi-vesicle formation has been the subject of ongoing discussions. Here, the discussion on the putative terminator/effector functions has been enlarged to include Arf GAP members involved in the formation of clathrin-coated vesicles. ACAP1, whose role has been studied extensively, enhances the recycling of endocytosed proteins to the plasma membrane. Importantly, this positive role appears to be an overall reflection of both the terminator and effector activities attributed to ACAP1. Other Arf GAP subtypes have also been suggested to possess both terminator and effector activities. Interestingly, while most Arf GAP proteins regulate membrane trafficking by acting as facilitators, a few Arf GAP subtypes act as inhibitors.

A spatial focusing model for G protein signals. Regulator of G protein signaling (RGS) protien-mediated kinetic scaffolding.

PubMed

Zhong, Huailing; Wade, Susan M; Woolf, Peter J; Linderman, Jennifer J; Traynor, John R; Neubig, Richard R

2003-02-28

Regulators of G protein signaling (RGS) are GTPase-accelerating proteins (GAPs), which can inhibit heterotrimeric G protein pathways. In this study, we provide experimental and theoretical evidence that high concentrations of receptors (as at a synapse) can lead to saturation of GDP-GTP exchange making GTP hydrolysis rate-limiting. This results in local depletion of inactive heterotrimeric G-GDP, which is reversed by RGS GAP activity. Thus, RGS enhances receptor-mediated G protein activation even as it deactivates the G protein. Evidence supporting this model includes a GTP-dependent enhancement of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to G(i) by RGS. The RGS domain of RGS4 is sufficient for this, not requiring the NH(2)- or COOH-terminal extensions. Furthermore, a kinetic model including only the GAP activity of RGS replicates the GTP-dependent enhancement of GTPgammaS binding observed experimentally. Finally in a Monte Carlo model, this mechanism results in a dramatic "spatial focusing" of active G protein. Near the receptor, G protein activity is maintained even with RGS due to the ability of RGS to reduce depletion of local Galpha-GDP levels permitting rapid recoupling to receptor and maintained G protein activation near the receptor. In contrast, distant signals are suppressed by the RGS, since Galpha-GDP is not depleted there. Thus, a novel RGS-mediated "kinetic scaffolding" mechanism is proposed which narrows the spatial range of active G protein around a cluster of receptors limiting the spill-over of G protein signals to more distant effector molecules, thus enhancing the specificity of G(i) protein signals.

In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

PubMed Central

Cooper, J A; Kashishian, A

1993-01-01

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8382774

The identification of protein domains that mediate functional interactions between Rab-GTPases and RabGAPs using 3D protein modeling.

PubMed

Davie, Jeremiah J; Faitar, Silviu L

2017-01-01

Currently, time-consuming serial in vitro experimentation involving immunocytochemistry or radiolabeled materials is required to identify which of the numerous Rab-GTPases (Rab) and Rab-GTPase activating proteins (RabGAP) are capable of functional interactions. These interactions are essential for numerous cellular functions, and in silico methods of reducing in vitro trial and error would accelerate the pace of research in cell biology. We have utilized a combination of three-dimensional protein modeling and protein bioinformatics to identify domains present in Rab proteins that are predictive of their functional interaction with a specific RabGAP. The RabF2 and RabSF1 domains appear to play functional roles in mediating the interaction between Rabs and RabGAPs. Moreover, the RabSF1 domain can be used to make in silico predictions of functional Rab/RabGAP pairs. This method is expected to be a broadly applicable tool for predicting protein-protein interactions where existing crystal structures for homologs of the proteins of interest are available.

Interactions of phosphatidylinositol kinase, GTPase-activating protein (GAP), and GAP-associated proteins with the colony-stimulating factor 1 receptor.

PubMed Central

Reedijk, M; Liu, X Q; Pawson, T

1990-01-01

The interactions of the macrophage colony-stimulating factor 1 (CSF-1) receptor with potential targets were investigated after ligand stimulation either of mouse macrophages or of fibroblasts that ectopically express mouse CSF-1 receptors. In Rat-2 cells expressing the mouse CSF-1 receptor, full activation of the receptor and cellular transformation require exogenous CSF-1, whereas NIH 3T3 cells expressing mouse c-fms are transformed by autocrine stimulation. Activated CSF-1 receptors physically associate with a phosphatidylinositol (PI) 3'-kinase. A mutant CSF-1 receptor with a deletion of the kinase insert region was deficient in its ability to bind functional PI 3'-kinase and to induce PI 3'-kinase activity precipitable with antiphosphotyrosine antibodies. In fibroblasts, CSF-1 stimulation also induced the phosphorylation of the GTPase-activating protein (GAP)-associated protein p62 on tyrosine, although GAP itself was a relatively poor substrate. In contrast to PI 3'-kinase association, phosphorylation of p62 and GAP was not markedly affected by deletion of the kinase insert region. These results indicate that the kinase insert region selectively enhances the CSF-1-dependent association of the CSF-1 receptor with active PI 3'-kinase. The insert deletion mutant retains considerable transforming activity in NIH 3T3 cells (G. Taylor, M. Reedijk, V. Rothwell, L. Rohrschneider, and T. Pawson, EMBO J. 8:2029-2037, 1989). This mutant was more seriously impaired in Rat-2 cell transformation, although mutant-expressing Rat-2 cells still formed small colonies in soft agar in the presence of CSF-1. Therefore, phosphorylation of GAP and p62 through activation of the CSF-1 receptor does not result in full fibroblast transformation. The interaction between the CSF-1 receptor and PI 3'-kinase may contribute to c-fms fibroblast transformation and play a role in CSF-1-stimulated macrophages. Images PMID:2172781

Purification, crystallization and preliminary X-ray analysis of the inverse F-BAR domain of the human srGAP2 protein.

PubMed

Wang, Hongpeng; Zhang, Yan; Zhang, Zhenyi; Jin, Wei Lin; Wu, Geng

2014-01-01

Bin-Amphiphysin-Rvs (BAR) domain proteins play essential roles in diverse cellular processes by inducing membrane invaginations or membrane protrusions. Among the BAR superfamily, the `classical' BAR and Fes/CIP4 homology BAR (F-BAR) subfamilies of proteins usually promote membrane invaginations, whereas the inverse BAR (I-BAR) subfamily generally incur membrane protrusions. Despite possessing an N-terminal F-BAR domain, the srGAP2 protein regulates neurite outgrowth and neuronal migration by causing membrane protrusions reminiscent of the activity of I-BAR domain proteins. In this study, the inverse F-BAR (IF-BAR) domain of human srGAP2 was overexpressed, purified and crystallized. The crystals of the srGAP2 IF-BAR domain protein diffracted to 3.50 Å resolution and belonged to space group P2(1). These results will facilitate further structural determination of the srGAP2 IF-BAR domain and the ultimate elucidation of its peculiar behaviour of inducing membrane protrusions rather than membrane invaginations.

Co-activation of RanGTPase and inhibition of GTP dissociation by Ran-GTP binding protein RanBP1.

PubMed Central

Bischoff, F R; Krebber, H; Smirnova, E; Dong, W; Ponstingl, H

1995-01-01

RCC1 (the regulator of chromosome condensation) stimulates guanine nucleotide dissociation on the Ras-related nuclear protein Ran. Both polypeptides are components of a regulatory pathway that has been implicated in regulating DNA replication, onset of and exit from mitosis, mRNA processing and transport, and import of proteins into the nucleus. In a search for further members of the RCC1-Ran signal pathway, we have identified proteins of 23, 45 and 300 kDa which tightly bind to Ran-GTP but not Ran-GDP. The purified soluble 23 kDa Ran binding protein RanBP1 does not activate RanGTPase, but increases GTP hydrolysis induced by the RanGTPase-activating protein RanGAP1 by an order of magnitude. In the absence of RanGAP, it strongly inhibits RCC1-induced exchange of Ran-bound GTP. In addition, it forms a stable complex with nucleotide-free RCC1-Ran. With these properties, it differs markedly from guanine diphosphate dissociation inhibitors which preferentially prevent the exchange of protein-bound GDP and in some cases were shown to inhibit GAP-induced GTP hydrolysis. RanBP1 is the first member of a new class of proteins regulating the binding and hydrolysis of GTP by Ras-related proteins. Images PMID:7882974

Gap junction blockage promotes cadmium-induced apoptosis in BRL 3A derived from Buffalo rat liver cells.

PubMed

Hu, Di; Zou, Hui; Han, Tao; Xie, Junze; Dai, Nannan; Zhuo, Liling; Gu, Jianhong; Bian, Jianchun; Yuan, Yan; Liu, Xuezhong; Liu, Zongping

2016-03-01

Gap junctions mediate direct communication between cells; however, toxicological cascade triggered by nonessential metals can abrogate cellular signaling mediated by gap junctions. Although cadmium (Cd) is known to induce apoptosis in organs and tissues, the mechanisms that underlie gap junction activity in Cd-induced apoptosis in BRL 3A rat liver cells has yet to be established. In this study, we showed that Cd treatment decreased the cell index (a measure of cellular electrical impedance) in BRL 3A cells. Mechanistically, we found that Cd exposure decreased expression of connexin 43 (Cx43), increased expression of p-Cx43 and elevated intracellular free Ca(2+) concentration, corresponding to a decrease in gap junctional intercellular communication. Gap junction blockage pretreatment with 18β-glycyrrhizic acid (GA) promoted Cd-induced apoptosis, involving changes in expression of Bax, Bcl-2, caspase-3 and the mitochondrial transmembrane electrical potential (Δψm). Additionally, GA was found to enhance ERK and p38 activation during Cd-induced activation of mitogen-activated protein kinases, but had no significant effect on JNK activation. Our results indicated the apoptosis-related proteins and the ERK and p38 signaling pathways may participate in gap junction blockage promoting Cd-induced apoptosis in BRL 3A cells.

ELMO Domains, Evolutionary and Functional Characterization of a Novel GTPase-activating Protein (GAP) Domain for Arf Protein Family GTPases*

PubMed Central

East, Michael P.; Bowzard, J. Bradford; Dacks, Joel B.; Kahn, Richard A.

2012-01-01

The human family of ELMO domain-containing proteins (ELMODs) consists of six members and is defined by the presence of the ELMO domain. Within this family are two subclassifications of proteins, based on primary sequence conservation, protein size, and domain architecture, deemed ELMOD and ELMO. In this study, we used homology searching and phylogenetics to identify ELMOD family homologs in genomes from across eukaryotic diversity. This demonstrated not only that the protein family is ancient but also that ELMOs are potentially restricted to the supergroup Opisthokonta (Metazoa and Fungi), whereas proteins with the ELMOD organization are found in diverse eukaryotes and thus were likely the form present in the last eukaryotic common ancestor. The segregation of the ELMO clade from the larger ELMOD group is consistent with their contrasting functions as unconventional Rac1 guanine nucleotide exchange factors and the Arf family GTPase-activating proteins, respectively. We used unbiased, phylogenetic sorting and sequence alignments to identify the most highly conserved residues within the ELMO domain to identify a putative GAP domain within the ELMODs. Three independent but complementary assays were used to provide an initial characterization of this domain. We identified a highly conserved arginine residue critical for both the biochemical and cellular GAP activity of ELMODs. We also provide initial evidence of the function of human ELMOD1 as an Arf family GAP at the Golgi. These findings provide the basis for the future study of the ELMOD family of proteins and a new avenue for the study of Arf family GTPases. PMID:23014990

Structure and function of gap junction proteins: role of gap junction proteins in embryonic heart development.

PubMed

Ahir, Bhavesh K; Pratten, Margaret K

2014-01-01

Intercellular (cell-to-cell) communication is a crucial and complex mechanism during embryonic heart development. In the cardiovascular system, the beating of the heart is a dynamic and key regulatory process, which is functionally regulated by the coordinated spread of electrical activity through heart muscle cells. Heart tissues are composed of individual cells, each bearing specialized cell surface membrane structures called gap junctions that permit the intercellular exchange of ions and low molecular weight molecules. Gap junction channels are essential in normal heart function and they assist in the mediated spread of electrical impulses that stimulate synchronized contraction (via an electrical syncytium) of cardiac tissues. This present review describes the current knowledge of gap junction biology. In the first part, we summarise some relevant biochemical and physiological properties of gap junction proteins, including their structure and function. In the second part, we review the current evidence demonstrating the role of gap junction proteins in embryonic development with particular reference to those involved in embryonic heart development. Genetics and transgenic animal studies of gap junction protein function in embryonic heart development are considered and the alteration/disruption of gap junction intercellular communication which may lead to abnormal heart development is also discussed.

Molecular and Behavioral Changes Associated with Adult Hippocampus-Specific SynGAP1 Knockout

ERIC Educational Resources Information Center

Muhia, Mary; Willadt, Silvia; Yee, Benjamin K.; Feldon, Joram; Paterna, Jean-Charles; Schwendener, Severin; Vogt, Kaspar; Kennedy, Mary B.; Knuesel, Irene

2012-01-01

The synaptic Ras/Rap-GTPase-activating protein (SynGAP1) plays a unique role in regulating specific downstream intracellular events in response to N-methyl-D-aspartate receptor (NMDAR) activation. Constitutive heterozygous loss of SynGAP1 disrupts NMDAR-mediated physiological and behavioral processes, but the disruptions might be of developmental…

Evolution and variability of Solanum RanGAP2, a cofactor in the incompatible interaction between the resistance protein GPA2 and the Globodera pallida effector Gp-RBP-1.

PubMed

Carpentier, Jean; Grenier, Eric; Esquibet, Magalie; Hamel, Louis-Philippe; Moffett, Peter; Manzanares-Dauleux, Maria J; Kerlan, Marie-Claire

2013-04-19

The Ran GTPase Activating Protein 2 (RanGAP2) was first described as a regulator of mitosis and nucleocytoplasmic trafficking. It was then found to interact with the Coiled-Coil domain of the Rx and GPA2 resistance proteins, which confer resistance to Potato Virus X (PVX) and potato cyst nematode Globodera pallida, respectively. RanGAP2 is thought to mediate recognition of the avirulence protein GP-RBP-1 by GPA2. However, the Gpa2-induced hypersensitive response appears to be relatively weak and Gpa2 is limited in terms of spectrum of efficiency as it is effective against only two nematode populations. While functional and evolutionary analyses of Gp-Rbp-1 and Gpa2 identified key residues in both the resistance and avirulence proteins that are involved in recognition determination, whether variation in RanGAP2 also plays a role in pathogen recognition has not been investigated. We amplified a total of 147 RanGAP2 sequences from 55 accessions belonging to 18 different di-and tetraploid Solanum species from the section Petota. Among the newly identified sequences, 133 haplotypes were obtained and 19.1% of the nucleotide sites were found to be polymorphic. The observed intra-specific nucleotide diversity ranges from 0.1 to 1.3%. Analysis of the selection pressures acting on RanGAP2 suggests that this gene evolved mainly under purifying selection. Nonetheless, we identified polymorphic positions in the protein sequence at the intra-specific level, which could modulate the activity of RanGAP2. Two polymorphic sites and a three amino-acid deletion in RanGAP2 were found to affect the timing and intensity of the Gpa2-induced hypersensitive response to avirulent GP-RBP-1 variants even though they did not confer any gain of recognition of virulent GP-RBP-1 variants. Our results highlight how a resistance gene co-factor can manage in terms of evolution both an established role as a cell housekeeping gene and an implication in plant parasite interactions. StRanGAP2 gene appears to evolve under purifying selection. Its variability does not seem to influence the specificity of GPA2 recognition but is able to modulate this activity by enhancing the defence response. It seems therefore that the interaction with the plant resistance protein GPA2 (and/or Rx) rather than with the nematode effector was the major force in the evolution of the RanGAP2 locus in potato. From a mechanistic point of view these results are in accordance with a physical interaction of RanGAP2 with GPA2 and suggest that RBP-1 would rather bind the RanGAP2-GPA2 complex than the RanGAP2 protein alone.

Increase of gap junction activities in SW480 human colorectal cancer cells.

PubMed

Bigelow, Kristina; Nguyen, Thu A

2014-07-09

Colorectal cancer is one of the most common cancers in the United States with an early detection rate of only 39%. Colorectal cancer cells along with other cancer cells exhibit many deficiencies in cell-to-cell communication, particularly gap junctional intercellular communication (GJIC). GJIC has been reported to diminish as cancer cells progress. Gap junctions are intercellular channels composed of connexin proteins, which mediate the direct passage of small molecules from one cell to the next. They are involved in the regulation of the cell cycle, cell differentiation, and cell signaling. Since the regulation of gap junctions is lost in colorectal cancer cells, the goal of this study is to determine the effect of GJIC restoration in colorectal cancer cells. Gap Junction Activity Assay and protein analysis were performed to evaluate the effects of overexpression of connexin 43 (Cx43) and treatment of PQ1, a small molecule, on GJIC. Overexpression of Cx43 in SW480 colorectal cancer cells causes a 6-fold increase of gap junction activity compared to control. This suggests that overexpressing Cx43 can restore GJIC. Furthermore, small molecule like PQ1 directly targeting gap junction channel was used to increase GJIC. Gap junction enhancers, PQ1, at 200 nM showed a 4-fold increase of gap junction activity in SW480 cells. A shift from the P0 to the P2 isoform of Cx43 was seen after 1 hour treatment with 200 nM PQ1. Overexpression of Cx43 and treatment of PQ1 can directly increase gap junction activity. The findings provide an important implication in which restoration of gap junction activity can be targeted for drug development.

The basic helix-loop-helix differentiation factor Nex1/MATH-2 functions as a key activator of the GAP-43 gene

PubMed Central

Uittenbogaard, Martine; Martinka, Debra L.; Chiaramello, Anne

2006-01-01

Nex1/MATH-2 is a neurogenic basic Helix-Loop-Helix (bHLH) transcription factor that belongs to the NeuroD subfamily. Its expression parallels that of the GAP-43 gene and peaks during brain development, when neurite outgrowth and synaptogenesis are highly active. We previously observed a direct correlation between the levels of expression of Nex1 and GAP-43 proteins, which resulted in extensive neurite outgrowth and neuronal differentiation of PC12 cells in the absence of nerve growth factor. Since the GAP-43 gene is a target for bHLH regulation, we investigated whether Nex1 could regulate the activity of the GAP-43 promoter. We found that among the members of the NeuroD subfamily, Nex1 promoted maximal activity of the GAP-43 promoter. The Nex1-mediated activity is restricted to the conserved E1–E2 cluster located near the major transcription start sites. By electrophoretic mobility shift assay and site-directed mutagenesis, we showed that Nex1 binds as homodimers and that the E1 E-box is a high affinity binding site. We further found that Nex1 released the ME1 E-protein-mediated repression in a concentration dependent manner. Thus, the E1–E2 cluster has a dual function: it can mediate activation or repression depending on the interacting bHLH proteins. Finally, a series of N-terminal and C-terminal deletions revealed that Nex1 transcriptional activity is linked to two distinct transactivation domains, TAD1 and TAD2, with TAD1 being unique to Nex1. Together, our results suggest that Nex1 may engage in selective interactions with components of the core transcriptional machinery whose assembly is dictated by the architecture of the GAP-43 promoter and cellular environment. PMID:12562512

RUTBC1 Functions as a GTPase-activating Protein for Rab32/38 and Regulates Melanogenic Enzyme Trafficking in Melanocytes*

PubMed Central

Marubashi, Soujiro; Shimada, Hikaru; Fukuda, Mitsunori; Ohbayashi, Norihiko

2016-01-01

Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed as regulating the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. Like other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and a GTP-bound active form; the cycle is thought to be regulated by an activating enzyme, guanine nucleotide exchange factor (GEF), and an inactivating enzyme, GTPase-activating protein (GAP), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has ever been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated the involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes. We discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicate that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes. PMID:26620560

The yeast Arf-GAP Glo3p is required for the endocytic recycling of cell surface proteins.

PubMed

Kawada, Daiki; Kobayashi, Hiromu; Tomita, Tsuyoshi; Nakata, Eisuke; Nagano, Makoto; Siekhaus, Daria Elisabeth; Toshima, Junko Y; Toshima, Jiro

2015-01-01

Small GTP-binding proteins of the Ras superfamily play diverse roles in intracellular trafficking. Among them, the Rab, Arf, and Rho families function in successive steps of vesicle transport, in forming vesicles from donor membranes, directing vesicle trafficking toward target membranes and docking vesicles onto target membranes. These proteins act as molecular switches that are controlled by a cycle of GTP binding and hydrolysis regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). In this study we explored the role of GAPs in the regulation of the endocytic pathway using fluorescently labeled yeast mating pheromone α-factor. Among 25 non-essential GAP mutants, we found that deletion of the GLO3 gene, encoding Arf-GAP protein, caused defective internalization of fluorescently labeled α-factor. Quantitative analysis revealed that glo3Δ cells show defective α-factor binding to the cell surface. Interestingly, Ste2p, the α-factor receptor, was mis-localized from the plasma membrane to the vacuole in glo3Δ cells. Domain deletion mutants of Glo3p revealed that a GAP-independent function, as well as the GAP activity, of Glo3p is important for both α-factor binding and Ste2p localization at the cell surface. Additionally, we found that deletion of the GLO3 gene affects the size and number of Arf1p-residing Golgi compartments and causes a defect in transport from the TGN to the plasma membrane. Furthermore, we demonstrated that glo3Δ cells were defective in the late endosome-to-TGN transport pathway, but not in the early endosome-to-TGN transport pathway. These findings suggest novel roles for Arf-GAP Glo3p in endocytic recycling of cell surface proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

Melatonin improves neuroplasticity by upregulating the growth-associated protein-43 (GAP-43) and NMDAR postsynaptic density-95 (PSD-95) proteins in cultured neurons exposed to glutamate excitotoxicity and in rats subjected to transient focal cerebral ischemia even during a long-term recovery period.

PubMed

Juan, Wei-Sheng; Huang, Sheng-Yang; Chang, Che-Chao; Hung, Yu-Chang; Lin, Yu-Wen; Chen, Tsung-Ying; Lee, Ai-Hua; Lee, Ai-Chiang; Wu, Tian-Shung; Lee, E-Jian

2014-03-01

Recent evidence shows that the NMDAR postsynaptic density-95 (PSD-95), growth-associated protein-43 (GAP-43), and matrix metalloproteinase-9 (MMP-9) protein enhance neuroplasticity at the subacute stage of stroke. Here, we evaluated whether melatonin would modulate the PSD-95, GAP-43, and MMP-9 proteins in cultured neurons exposed to glutamate excitotoxicity and in rats subjected to experimental stroke. Adult male Sprague-Dawley rats were treated with melatonin (5 mg/kg) or vehicle at reperfusion onset after transient occlusion of the right middle cerebral artery (tMCAO) for 90 min. Animals were euthanized for Western immunoblot analyses for the PSD-95 and GAP-43 proteins and gelatin zymography for the MMP-9 activity at 7 days postinsult. Another set of animals was sacrificed for histologic and Golgi-Cox-impregnated sections at 28 days postinsult. In cultured neurons exposed to glutamate excitotoxicity, melatonin significantly upregulated the GAP-43 and PSD-95 expressions and improved dendritic aborizations (P<0.05, respectively). Relative to controls, melatonin-treated stroke animals caused a significant improvement in GAP-43 and PSD-95 expressions as well as the MMP-9 activity in the ischemic brain (P<0.05). Consequently, melatonin also significantly promoted the dendritic spine density and reduced infarction in the ischemic brain, and improved neurobehaviors as well at 28 days postinsult (P<0.05, respectively). Together, melatonin upregulates GAP-43, PSD-95, and MMP-9 proteins, which likely accounts for its actions to improve neuroplasticity in cultured neurons exposed to glutamate excitotoxicity and to enhance long-term neuroprotection, neuroplasticity, and brain remodeling in stroke rats. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Internal amino acids promote Gap1 permease ubiquitylation via TORC1/Npr1/14-3-3-dependent control of the Bul arrestin-like adaptors.

PubMed

Merhi, Ahmad; André, Bruno

2012-11-01

Ubiquitylation of many plasma membrane proteins promotes their endocytosis followed by degradation in the lysosome. The yeast general amino acid permease, Gap1, is ubiquitylated and downregulated when a good nitrogen source like ammonium is provided to cells growing on a poor nitrogen source. This ubiquitylation requires the Rsp5 ubiquitin ligase and the redundant arrestin-like Bul1 and Bul2 adaptors. Previous studies have shown that Gap1 ubiquitylation involves the TORC1 kinase complex, which inhibits the Sit4 phosphatase. This causes inactivation of the protein kinase Npr1, which protects Gap1 against ubiquitylation. However, the mechanisms inducing Gap1 ubiquitylation after Npr1 inactivation remain unknown. We here show that on a poor nitrogen source, the Bul adaptors are phosphorylated in an Npr1-dependent manner and bound to 14-3-3 proteins that protect Gap1 against downregulation. After ammonium is added and converted to amino acids, the Bul proteins are dephosphorylated, dissociate from the 14-3-3 proteins, and undergo ubiquitylation. Furthermore, dephosphorylation of Bul requires the Sit4 phosphatase, which is essential to Gap1 downregulation. The data support the emerging concept that permease ubiquitylation results from activation of the arrestin-like adaptors of the Rsp5 ubiquitin ligase, this coinciding with their dephosphorylation, dissociation from the inhibitory 14-3-3 proteins, and ubiquitylation.

A model for regulation by SynGAP-α1 of binding of synaptic proteins to PDZ-domain 'Slots' in the postsynaptic density

PubMed Central

Walkup, Ward G; Mastro, Tara L; Schenker, Leslie T; Vielmetter, Jost; Hu, Rebecca; Iancu, Ariella; Reghunathan, Meera; Bannon, Barry Dylan; Kennedy, Mary B

2016-01-01

SynGAP is a Ras/Rap GTPase-activating protein (GAP) that is a major constituent of postsynaptic densities (PSDs) from mammalian forebrain. Its α1 isoform binds to all three PDZ (PSD-95, Discs-large, ZO-1) domains of PSD-95, the principal PSD scaffold, and can occupy as many as 15% of these PDZ domains. We present evidence that synGAP-α1 regulates the composition of the PSD by restricting binding to the PDZ domains of PSD-95. We show that phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and Polo-like kinase-2 (PLK2) decreases its affinity for the PDZ domains by several fold, which would free PDZ domains for occupancy by other proteins. Finally, we show that three critical postsynaptic signaling proteins that bind to the PDZ domains of PSD-95 are present in higher concentration in PSDs isolated from mice with a heterozygous deletion of synGAP. DOI: http://dx.doi.org/10.7554/eLife.16813.001 PMID:27623146

Spatio-temporal regulation of connexin43 phosphorylation and gap junction dynamics.

PubMed

Solan, Joell L; Lampe, Paul D

2018-01-01

Gap junctions are specialized membrane domains containing tens to thousands of intercellular channels. These channels permit exchange of small molecules (<1000Da) including ions, amino acids, nucleotides, metabolites and secondary messengers (e.g., calcium, glucose, cAMP, cGMP, IP 3 ) between cells. The common reductionist view of these structures is that they are composed entirely of integral membrane proteins encoded by the 21 member connexin human gene family. However, it is clear that the normal physiological function of this structure requires interaction and regulation by a variety of proteins, especially kinases. Phosphorylation is capable of directly modulating connexin channel function but the most dramatic effects on gap junction activity occur via the organization of the gap junction structures themselves. This is a direct result of the short half-life of the primary gap junction protein, connexin, which requires them to be constantly assembled, remodeled and turned over. The biological consequences of this remodeling are well illustrated during cardiac ischemia, a process wherein gap junctions are disassembled and remodeled resulting in arrhythmia and ultimately heart failure. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve. Copyright © 2017 Elsevier B.V. All rights reserved.

RhoA GTPase inhibition organizes contraction during epithelial morphogenesis

PubMed Central

Mason, Frank M.; Xie, Shicong; Vasquez, Claudia G.; Tworoger, Michael

2016-01-01

During morphogenesis, contraction of the actomyosin cytoskeleton within individual cells drives cell shape changes that fold tissues. Coordination of cytoskeletal contractility is mediated by regulating RhoA GTPase activity. Guanine nucleotide exchange factors (GEFs) activate and GTPase-activating proteins (GAPs) inhibit RhoA activity. Most studies of tissue folding, including apical constriction, have focused on how RhoA is activated by GEFs to promote cell contractility, with little investigation as to how GAPs may be important. Here, we identify a critical role for a RhoA GAP, Cumberland GAP (C-GAP), which coordinates with a RhoA GEF, RhoGEF2, to organize spatiotemporal contractility during Drosophila melanogaster apical constriction. C-GAP spatially restricts RhoA pathway activity to a central position in the apical cortex. RhoGEF2 pulses precede myosin, and C-GAP is required for pulsation, suggesting that contractile pulses result from RhoA activity cycling. Finally, C-GAP expression level influences the transition from reversible to irreversible cell shape change, which defines the onset of tissue shape change. Our data demonstrate that RhoA activity cycling and modulating the ratio of RhoGEF2 to C-GAP are required for tissue folding. PMID:27551058

EHB1 and AGD12, two calcium-dependent proteins affect gravitropism antagonistically in Arabidopsis thaliana.

PubMed

Dümmer, Michaela; Michalski, Christian; Essen, Lars-Oliver; Rath, Magnus; Galland, Paul; Forreiter, Christoph

2016-11-01

The ADP-RIBOSYLATION FACTOR GTPase-ACTIVATING PROTEIN (AGD) 12, a member of the ARF-GAP protein family, affects gravitropism in Arabidopsis thaliana. A loss-of-function mutant lacking AGD12 displayed diminished gravitropism in roots and hypocotyls indicating that both organs are affected by this regulator. AGD12 is structurally related to ENHANCED BENDING (EHB) 1, previously described as a negative effector of gravitropism. In contrast to agd12 mutants, ehb1 loss-of function seedlings displayed enhanced gravitropic bending. While EHB1 and AGD12 both possess a C-terminal C2/CaLB-domain, EHB1 lacks the N-terminal ARF-GAP domain present in AGD12. Subcellular localization analysis using Brefeldin A indicated that both proteins are elements of the trans Golgi network. Physiological analyses provided evidence that gravitropic signaling might operate via an antagonistic interaction of ARF-GAP (AGD12) and EHB1 in their Ca 2+ -activated states. Copyright © 2016 Elsevier GmbH. All rights reserved.

Site-directed Mutagenesis Shows the Significance of Interactions with Phospholipids and the G-protein OsYchF1 for the Physiological Functions of the Rice GTPase-activating Protein 1 (OsGAP1).

PubMed

Yung, Yuk-Lin; Cheung, Ming-Yan; Miao, Rui; Fong, Yu-Hang; Li, Kwan-Pok; Yu, Mei-Hui; Chye, Mee-Len; Wong, Kam-Bo; Lam, Hon-Ming

2015-09-25

The C2 domain is one of the most diverse phospholipid-binding domains mediating cellular signaling. One group of C2-domain proteins are plant-specific and are characterized by their small sizes and simple structures. We have previously reported that a member of this group, OsGAP1, is able to alleviate salt stress and stimulate defense responses, and bind to both phospholipids and an unconventional G-protein, OsYchF1. Here we solved the crystal structure of OsGAP1 to a resolution of 1.63 Å. Using site-directed mutagenesis, we successfully differentiated between the clusters of surface residues that are required for binding to phospholipids versus OsYchF1, which, in turn, is critical for its role in stimulating defense responses. On the other hand, the ability to alleviate salt stress by OsGAP1 is dependent only on its ability to bind OsYchF1 and is independent of its phospholipid-binding activity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Phosphatidic acid binding inhibits RGS1 activity to affect specific signaling pathways in Arabidopsis.

PubMed

Roy Choudhury, Swarup; Pandey, Sona

2017-05-01

Modulation of the active versus inactive forms of the Gα protein is critical for the signaling processes mediated by the heterotrimeric G-protein complex. We have recently established that in Arabidopsis, the regulator of G-protein signaling (RGS1) protein and a lipid-hydrolyzing enzyme, phospholipase Dα1 (PLDα1), both act as GTPase-activity accelerating proteins (GAPs) for the Gα protein to attenuate its activity. RGS1 and PLDα1 interact with each other, and RGS1 inhibits the activity of PLDα1 during regulation of a subset of responses. In this study, we present evidence that this regulation is bidirectional. Phosphatidic acid (PA), a second messenger typically derived from the lipid-hydrolyzing activity of PLDα1, is a molecular target of RGS1. PA binds and inhibits the GAP activity of RGS1. A conserved lysine residue in RGS1 (Lys 259 ) is directly involved in RGS1-PA binding. Introduction of this RGS1 protein variant in the rgs1 mutant background makes plants hypersensitive to a subset of abscisic acid-mediated responses. Our data point to the existence of negative feedback loops between these two regulatory proteins that precisely modulate the level of active Gα, consequently generating a highly controlled signal-response output. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor.

PubMed

Nie, Zhongzhen; Hirsch, Dianne S; Luo, Ruibai; Jian, Xiaoying; Stauffer, Stacey; Cremesti, Aida; Andrade, Josefa; Lebowitz, Jacob; Marino, Michael; Ahvazi, Bijan; Hinshaw, Jenny E; Randazzo, Paul A

2006-01-24

Arf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1. ASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect. The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.

The RhoGAP activity of CYK-4/MgcRacGAP functions non-canonically by promoting RhoA activation during cytokinesis

PubMed Central

Zhang, Donglei; Glotzer, Michael

2015-01-01

Cytokinesis requires activation of the GTPase RhoA. ECT-2, the exchange factor responsible for RhoA activation, is regulated to ensure spatiotemporal control of contractile ring assembly. Centralspindlin, composed of the Rho family GTPase-activating protein (RhoGAP) MgcRacGAP/CYK-4 and the kinesin MKLP1/ZEN-4, is known to activate ECT-2, but the underlying mechanism is not understood. We report that ECT-2-mediated RhoA activation depends on the ability of CYK-4 to localize to the plasma membrane, bind RhoA, and promote GTP hydrolysis by RhoA. Defects resulting from loss of CYK-4 RhoGAP activity can be rescued by activating mutations in ECT-2 or depletion of RGA-3/4, which functions as a conventional RhoGAP for RhoA. Consistent with CYK-4 RhoGAP activity contributing to GEF activation, the catalytic domains of CYK-4 and ECT-2 directly interact. Thus, counterintuitively, CYK-4 RhoGAP activity promotes RhoA activation. We propose that the most active form of the cytokinetic RhoGEF involves complex formation between ECT-2, centralspindlin and RhoA. DOI: http://dx.doi.org/10.7554/eLife.08898.001 PMID:26252513

An extracellular-matrix-specific GEF-GAP interaction regulates Rho GTPase crosstalk for 3D collagen migration.

PubMed

Kutys, Matthew L; Yamada, Kenneth M

2014-09-01

Rho-family GTPases govern distinct types of cell migration on different extracellular matrix proteins in tissue culture or three-dimensional (3D) matrices. We searched for mechanisms selectively regulating 3D cell migration in different matrix environments and discovered a form of Cdc42-RhoA crosstalk governing cell migration through a specific pair of GTPase activator and inhibitor molecules. We first identified βPix, a guanine nucleotide exchange factor (GEF), as a specific regulator of migration in 3D collagen using an affinity-precipitation-based GEF screen. Knockdown of βPix specifically blocks cell migration in fibrillar collagen microenvironments, leading to hyperactive cellular protrusion accompanied by increased collagen matrix contraction. Live FRET imaging and RNAi knockdown linked this βPix knockdown phenotype to loss of polarized Cdc42 but not Rac1 activity, accompanied by enhanced, de-localized RhoA activity. Mechanistically, collagen phospho-regulates βPix, leading to its association with srGAP1, a GTPase-activating protein (GAP), needed to suppress RhoA activity. Our results reveal a matrix-specific pathway controlling migration involving a GEF-GAP interaction of βPix with srGAP1 that is critical for maintaining suppressive crosstalk between Cdc42 and RhoA during 3D collagen migration.

A domain unique to plant RanGAP is responsible for its targeting to the plant nuclear rim

PubMed Central

Rose, Annkatrin; Meier, Iris

2001-01-01

Ran is a small signaling GTPase that is involved in nucleocytoplasmic transport. Two additional functions of animal Ran in the formation of spindle asters and the reassembly of the nuclear envelope in mitotic cells have been recently reported. In contrast to Ras or Rho, Ran is not associated with membranes. Instead, the spatial sequestering of its accessory proteins, the Ran GTPase-activating protein RanGAP and the nucleotide exchange factor RCC1, appears to define the local concentration of RanGTP vs. RanGDP involved in signaling. Mammalian RanGAP is bound to the nuclear pore by a mechanism involving the attachment of small ubiquitin-related modifier protein (SUMO) to its C terminus and the subsequent binding of the SUMOylated domain to the nucleoporin Nup358. Here we show that plant RanGAP utilizes a different mechanism for nuclear envelope association, involving a novel targeting domain that appears to be unique to plants. The N-terminal WPP domain is highly conserved among plant RanGAPs and the small, plant-specific nuclear envelope-associated protein MAF1, but not present in yeast or animal RanGAP. Confocal laser scanning microscopy of green fluorescent protein (GFP) fusion proteins showed that it is necessary for RanGAP targeting and sufficient to target the heterologous protein GFP to the plant nuclear rim. The highly conserved tryptophan and proline residues of the WPP motif are necessary for its function. The 110-aa WPP domain is the first nuclear-envelope targeting domain identified in plants. Its fundamental difference to its mammalian counterpart implies that different mechanisms have evolved in plants and animals to anchor RanGAP at the nuclear surface. PMID:11752475

The GAP arginine finger movement into the catalytic site of Ras increases the activation entropy

PubMed Central

Kötting, Carsten; Kallenbach, Angela; Suveyzdis, Yan; Wittinghofer, Alfred; Gerwert, Klaus

2008-01-01

Members of the Ras superfamily of small G proteins play key roles in signal transduction pathways, which they control by GTP hydrolysis. They are regulated by GTPase activating proteins (GAPs). Mutations that prevent hydrolysis cause severe diseases including cancer. A highly conserved “arginine finger” of GAP is a key residue. Here, we monitor the GTPase reaction of the Ras·RasGAP complex at high temporal and spatial resolution by time-resolved FTIR spectroscopy at 260 K. After triggering the reaction, we observe as the first step a movement of the switch-I region of Ras from the nonsignaling “off” to the signaling “on” state with a rate of 3 s−1. The next step is the movement of the “arginine finger” into the active site of Ras with a rate of k2 = 0.8 s−1. Once the arginine points into the binding pocket, cleavage of GTP is fast and the protein-bound Pi intermediate forms. The switch-I reversal to the “off” state, the release of Pi, and the movement of arginine back into an aqueous environment is observed simultaneously with k3 = 0.1 s−1, the rate-limiting step. Arrhenius plots for the partial reactions show that the activation energy for the cleavage reaction is lowered by favorable positive activation entropy. This seems to indicate that protein-bound structured water molecules are pushed by the “arginine finger” movement out of the binding pocket into the bulk water. The proposed mechanism shows how the high activation barrier for phosphoryl transfer can be reduced by splitting into partial reactions separated by a Pi-intermediate. PMID:18434546

A Conserved RhoGAP Limits M-phase Contractility and Coordinates with Microtubule Asters to Restrict Active RhoA to the Cell Equator During Cytokinesis

PubMed Central

Zanin, Esther; Desai, Arshad; Poser, Ina; Toyoda, Yusuke; Andree, Cordula; Moebius, Claudia; Bickle, Marc; Conradt, Barbara; Piekny, Alisa; Oegema, Karen

2014-01-01

SUMMARY During animal cell cytokinesis, the spindle directs contractile ring assembly by activating RhoA in a narrow equatorial zone. Rapid GTPase activating protein (GAP)-mediated inactivation (RhoA flux) is proposed to limit RhoA zone dimensions. Testing the significance of RhoA flux has been hampered by the fact that the GAP targeting RhoA is not known. Here, we identify M-phase GAP (MP-GAP) as the primary GAP targeting RhoA during mitosis/cytokinesis. MP-GAP inhibition caused excessive RhoA activation in M-phase leading to the uncontrolled formation of large cortical protrusions and late cytokinesis failure. RhoA zone width was broadened by attenuation of the centrosomal asters but was not affected by MP-GAP inhibition alone. Simultaneous aster attenuation and MP-GAP inhibition led to RhoA accumulation around the entire cell periphery. These results identify the major GAP restraining RhoA during cell division and delineate the relative contributions of RhoA flux and centrosomal asters in controlling RhoA zone dimensions. PMID:24012485

Neuronal ELAV proteins enhance mRNA stability by a PKCα-dependent pathway

PubMed Central

Pascale, Alessia; Amadio, Marialaura; Scapagnini, Giovanni; Lanni, Cristina; Racchi, Marco; Provenzani, Alessandro; Govoni, Stefano; Alkon, Daniel L.; Quattrone, Alessandro

2005-01-01

More than 1 in 20 human genes bear in the mRNA 3′ UTR a specific motif called the adenine- and uridine-rich element (ARE), which posttranscriptionally determines its expression in response to cell environmental signals. ELAV (embryonic lethal abnormal vision) proteins are the only known ARE-binding factors that are able to stabilize the bound mRNAs, thereby positively controlling gene expression. Here, we show that in human neuroblastoma SH-SY5Y cells, neuron-specific ELAV (nELAV) proteins (HuB, HuC, and HuD) are up-regulated and redistributed by 15 min of treatment with the activators of PKC phorbol esters and bryostatin-1. PKC stimulation also induces nELAV proteins to colocalize with the translocated PKCα isozyme preferentially on the cytoskeleton, with a concomitant increase of nELAV threonine phosphorylation. The same treatment promotes stabilization of growth-associated protein 43 (GAP-43) mRNA, a well known nELAV target, and induces an early increase in GAP-43 protein concentration, again only in the cytoskeletal cell fraction. Genetic or pharmacological inactivation of PKCα abolishes nELAV protein cytoskeletal up-regulation, GAP-43 mRNA stabilization, and GAP-43 protein increase, demonstrating the primary role of this specific PKC isozyme in the cascade of nELAV recruitment. Finally, in vivo PKC activation is associated with an up-regulation of nELAV proteins in the hippocampal rat brain. These findings suggest a model for gene expression regulation by nELAV proteins through a PKCα-dependent pathway that is relevant for the cellular programs in which ARE-mediated control plays a pivotal role. PMID:16099831

Specific Rab GTPase-activating proteins define the Shiga toxin and epidermal growth factor uptake pathways.

PubMed

Fuchs, Evelyn; Haas, Alexander K; Spooner, Robert A; Yoshimura, Shin-ichiro; Lord, J Michael; Barr, Francis A

2007-06-18

Rab family guanosine triphosphatases (GTPases) together with their regulators define specific pathways of membrane traffic within eukaryotic cells. In this study, we have investigated which Rab GTPase-activating proteins (GAPs) can interfere with the trafficking of Shiga toxin from the cell surface to the Golgi apparatus and studied transport of the epidermal growth factor (EGF) from the cell surface to endosomes. This screen identifies 6 (EVI5, RN-tre/USP6NL, TBC1D10A-C, and TBC1D17) of 39 predicted human Rab GAPs as specific regulators of Shiga toxin but not EGF uptake. We show that Rab43 is the target of RN-tre and is required for Shiga toxin uptake. In contrast, RabGAP-5, a Rab5 GAP, was unique among the GAPs tested and reduced the uptake of EGF but not Shiga toxin. These results suggest that Shiga toxin trafficking to the Golgi is a multistep process controlled by several Rab GAPs and their target Rabs and that this process is discrete from ligand-induced EGF receptor trafficking.

RabGAP22 Is Required for Defense to the Vascular Pathogen Verticillium longisporum and Contributes to Stomata Immunity

PubMed Central

Roos, Jonas; Bejai, Sarosh; Oide, Shinichi; Dixelius, Christina

2014-01-01

Verticillium longisporum is a soil-borne pathogen with a preference for plants within the family Brassicaceae. Following invasion of the roots, the fungus proliferates in the plant vascular system leading to stunted plant growth, chlorosis and premature senescence. RabGTPases have been demonstrated to play a crucial role in regulating multiple responses in plants. Here, we report on the identification and characterization of the Rab GTPase-activating protein RabGAP22 gene from Arabidopsis, as an activator of multiple components in the immune responses to V. longisporum. RabGAP22Pro:GUS transgenic lines showed GUS expression predominantly in root meristems, vascular tissues and stomata, whereas the RabGAP22 protein localized in the nucleus. Reduced RabGAP22 transcript levels in mutants of the brassinolide (BL) signaling gene BRI1-ASSOCIATED RECEPTOR KINASE 1, together with a reduction of fungal proliferation following BL pretreatment, suggested RabGAP22 to be involved in BL-mediated responses. Pull-down assays revealed SERINE:GLYOXYLATE AMINOTRANSFERASE (AGT1) as an interacting partner during V. longisporum infection and bimolecular fluorescence complementation (BiFC) showed the RabGAP22-AGT1 protein complex to be localized in the peroxisomes. Further, fungal-induced RabGAP22 expression was found to be associated with elevated endogenous levels of the plant hormones jasmonic acid (JA) and abscisic acid (ABA). An inadequate ABA response in rabgap22-1 mutants, coupled with a stomata-localized expression of RabGAP22 and impairment of guard cell closure in response to V. longisporum and Pseudomonas syringae, suggest that RabGAP22 has multiple roles in innate immunity. PMID:24505423

RUTBC1 Functions as a GTPase-activating Protein for Rab32/38 and Regulates Melanogenic Enzyme Trafficking in Melanocytes.

PubMed

Marubashi, Soujiro; Shimada, Hikaru; Fukuda, Mitsunori; Ohbayashi, Norihiko

2016-01-15

Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed as regulating the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. Like other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and a GTP-bound active form; the cycle is thought to be regulated by an activating enzyme, guanine nucleotide exchange factor (GEF), and an inactivating enzyme, GTPase-activating protein (GAP), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has ever been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated the involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes. We discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicate that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

General anesthetics have differential inhibitory effects on gap junction channels and hemichannels in astrocytes and neurons.

PubMed

Liu, Xinhe; Gangoso, Ester; Yi, Chenju; Jeanson, Tiffany; Kandelman, Stanislas; Mantz, Jean; Giaume, Christian

2016-04-01

Astrocytes represent a major non-neuronal cell population actively involved in brain functions and pathologies. They express a large amount of gap junction proteins that allow communication between adjacent glial cells and the formation of glial networks. In addition, these membrane proteins can also operate as hemichannels, through which "gliotransmitters" are released, and thus contribute to neuroglial interaction. There are now reports demonstrating that alterations of astroglial gap junction communication and/or hemichannel activity impact neuronal and synaptic activity. Two decades ago we reported that several general anesthetics inhibited gap junctions in primary cultures of astrocytes (Mantz et al., (1993) Anesthesiology 78(5):892-901). As there are increasing studies investigating neuroglial interactions in anesthetized mice, we here updated this previous study by employing acute cortical slices and by characterizing the effects of general anesthetics on both astroglial gap junctions and hemichannels. As hemichannel activity is not detected in cortical astrocytes under basal conditions, we treated acute slices with the endotoxin LPS or proinflammatory cytokines to induce hemichannel activity in astrocytes, which in turn activated neuronal hemichannels. We studied two extensively used anesthetics, propofol and ketamine, and the more recently developed dexmedetomidine. We report that these drugs have differential inhibitory effects on gap junctional communication and hemichannel activity in astrocytes when used in their respective, clinically relevant concentrations, and that dexmedetomidine appears to be the least effective on both channel functions. In addition, the three anesthetics have similar effects on neuronal hemichannels. Altogether, our observations may contribute to optimizing the selection of anesthetics for in vivo animal studies. © 2015 Wiley Periodicals, Inc.

Enhanced p122RhoGAP/DLC-1 Expression Can Be a Cause of Coronary Spasm

PubMed Central

Kinjo, Takahiko; Tanaka, Makoto; Osanai, Tomohiro; Shibutani, Shuji; Narita, Ikuyo; Tanno, Tomohiro; Nishizaki, Kimitaka; Ichikawa, Hiroaki; Kimura, Yoshihiro; Ishida, Yuji; Yokota, Takashi; Shimada, Michiko; Homma, Yoshimi; Tomita, Hirofumi; Okumura, Ken

2015-01-01

Background We previously showed that phospholipase C (PLC)-δ1 activity was enhanced by 3-fold in patients with coronary spastic angina (CSA). We also reported that p122Rho GTPase-activating protein/deleted in liver cancer-1 (p122RhoGAP/DLC-1) protein, which was discovered as a PLC-δ1 stimulator, was upregulated in CSA patients. We tested the hypothesis that p122RhoGAP/DLC-1 overexpression causes coronary spasm. Methods and Results We generated transgenic (TG) mice with vascular smooth muscle (VSM)-specific overexpression of p122RhoGAP/DLC-1. The gene and protein expressions of p122RhoGAP/DLC-1 were markedly increased in the aorta of homozygous TG mice. Stronger staining with anti-p122RhoGAP/DLC-1 in the coronary artery was found in TG than in WT mice. PLC activities in the plasma membrane fraction and the whole cell were enhanced by 1.43 and 2.38 times, respectively, in cultured aortic vascular smooth muscle cells from homozygous TG compared with those from WT mice. Immediately after ergometrine injection, ST-segment elevation was observed in 1 of 7 WT (14%), 6 of 7 heterozygous TG (84%), and 7 of 7 homozygous TG mice (100%) (p<0.05, WT versus TGs). In the isolated Langendorff hearts, coronary perfusion pressure was increased after ergometrine in TG, but not in WT mice, despite of the similar response to prostaglandin F2α between TG and WT mice (n = 5). Focal narrowing of the coronary artery after ergometrine was documented only in TG mice. Conclusions VSM-specific overexpression of p122RhoGAP/DLC-1 enhanced coronary vasomotility after ergometrine injection in mice, which is relevant to human CSA. PMID:26624289

Reduced expression of the NMDA receptor-interacting protein SynGAP causes behavioral abnormalities that model symptoms of Schizophrenia.

PubMed

Guo, Xiaochuan; Hamilton, Peter J; Reish, Nicholas J; Sweatt, J David; Miller, Courtney A; Rumbaugh, Gavin

2009-06-01

Abnormal function of NMDA receptors is believed to be a contributing factor to the pathophysiology of schizophrenia. NMDAR subunits and postsynaptic-interacting proteins of these channels are abnormally expressed in some patients with this illness. In mice, reduced NMDAR expression leads to behaviors analogous to symptoms of schizophrenia, but reports of animals with mutations in core postsynaptic density proteins having similar a phenotype have yet to be reported. Here we show that reduced expression of the neuronal RasGAP and NMDAR-associated protein, SynGAP, results in abnormal behaviors strikingly similar to that reported in mice with reduced NMDAR function. SynGAP mutant mice exhibited nonhabituating and persistent hyperactivity that was ameliorated by the antipsychotic clozapine. An NMDAR antagonist, MK-801, induced hyperactivity in normal mice but SynGAP mutants were less responsive, suggesting that NMDAR hypofunction contributes to this behavioral abnormality. SynGAP mutants exhibited enhanced startle reactivity and impaired sensory-motor gating. These mice also displayed a complete lack of social memory and a propensity toward social isolation. Finally, SynGAP mutants had deficits in cued fear conditioning and working memory, indicating abnormal function of circuits that control emotion and choice. Our results demonstrate that SynGAP mutant mice have gross neurological deficits similar to other mouse models of schizophrenia. Because SynGAP interacts with NMDARs, and the signaling activity of this protein is regulated by these channels, our data in dicate that SynGAP lies downstream of NMDARs and is a required intermediate for normal neural circuit function and behavior. Taken together, these data support the idea that schizophrenia may arise from abnormal signaling pathways that are mediated by NMDA receptors.

Through its F-BAR and RhoGAP domains, Rgd1p acts in different polarized growth processes in budding yeast

PubMed Central

Lefebvre, Fabien; Prouzet-Mauléon, Valérie; Vieillemard, Aurélie; Thoraval, Didier; Crouzet, Marc

2009-01-01

Protein domain architecture can be used to construct supramolecular structures, to carry out specific functions and to mediate signaling in prokaryotic and eukaryotic cells. The Rgd1p protein of budding yeast contains two domains with different functions in the cell: the F-BAR and RhoGAP domains. The F-BAR domain has been shown to interact with membrane phospholipids and is thought to induce or sense membrane curvature. The RhoGAP domain activates the GTP hydrolysis of two Rho GTPases, thereby regulating different cellular pathways. Specific molecular interactions with the F-BAR and RhoGAP domains, cell signaling and interplay between these domains may allow the Rgd1p protein to act in several different biological processes, all of which are required for polarized growth in yeast. PMID:19704907

Capsaicin-induced activation of ERK1/2 and its involvement in GAP-43 expression and CGRP depletion in organotypically cultured DRG neurons.

PubMed

Li, Yunfeng; Liu, Guixiang; Li, Hao; Xu, Youzheng; Zhang, Hong; Liu, Zhen

2013-04-01

Low concentrations of capsaicin (CAP) stimulate and high concentrations of CAP can be toxic to the primary sensory neurons of the dorsal root ganglion (DRG). CAP induces the phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in DRG neurons. The effect of the activation of ERK1/2 by different concentrations of CAP on growth-associated protein 43 (GAP-43) expression and calcitonin gene-related peptide (CGRP) depletion in DRG neurons remains unknown. In the present study, organotypic embryonic 15-day-old rat DRG explants were used to determine the effect of different concentrations of CAP on GAP-43 expression and CGRP depletion. The results showed that, compared to unstimulated control cultures, GAP-43 and pERK1/2 protein levels increased at a low concentration (2 μmol/L) of CAP and decreased at a higher concentration (10 μmol/L). The number of CGRP-immunoreactive (IR) migrating neurons also decreased in CAP-treated cultures. The increase in GAP-43 levels and CGRP depletion could be blocked by the administration of ERK1/2 inhibitor PD98059. The results of the present study imply that CAP at different concentrations has different effects on GAP-43 expression and CGRP depletion. These effects were involved in the activation of ERK1/2 in organotypically cultured DRG neurons stimulated with CAP. These data may provide new insights for further development potential therapeutic applications of CAP with moderate dose on neurogenic inflammation.

Alterations in gap junction connexin43/connexin45 ratio mediate a transition from quiescence to excitation in a mathematical model of the myometrium

PubMed Central

Sheldon, Rachel E.; Mashayamombe, Chipo; Shi, Shao-Qing; Garfield, Robert E.; Shmygol, Anatoly; Blanks, Andrew M.; van den Berg, Hugo A.

2014-01-01

The smooth muscle cells of the uterus contract in unison during delivery. These cells achieve coordinated activity via electrical connections called gap junctions which consist of aggregated connexin proteins such as connexin43 and connexin45. The density of gap junctions governs the excitability of the myometrium (among other factors). An increase in gap junction density occurs immediately prior to parturition. We extend a mathematical model of the myometrium by incorporating the voltage-dependence of gap junctions that has been demonstrated in the experimental literature. Two functional subtypes exist, corresponding to systems with predominantly connexin43 and predominantly connexin45, respectively. Our simulation results indicate that the gap junction protein connexin45 acts as a negative modulator of uterine excitability, and hence, activity. A network with a higher proportion of connexin45 relative to connexin43 is unable to excite every cell. Connexin45 has much more rapid gating kinetics than connexin43 which we show limits the maximum duration of a local burst of activity. We propose that this effect regulates the degree of synchronous excitation attained during a contraction. Our results support the hypothesis that as labour approaches, connexin45 is downregulated to allow action potentials to spread more readily through the myometrium. PMID:25401181

Spinal astrocyte gap junctions contribute to oxaliplatin-induced mechanical hypersensitivity.

PubMed

Yoon, Seo-Yeon; Robinson, Caleb R; Zhang, Haijun; Dougherty, Patrick M

2013-02-01

Spinal glial cells contribute to the development of many types of inflammatory and neuropathic pain. Here the contribution of spinal astrocytes and astrocyte gap junctions to oxaliplatin-induced mechanical hypersensitivity was explored. The expression of glial fibrillary acidic protein (GFAP) in spinal dorsal horn was significantly increased at day 7 but recovered at day 14 after oxaliplatin treatment, suggesting a transient activation of spinal astrocytes by chemotherapy. Astrocyte-specific gap junction protein connexin 43 (Cx43) was significantly increased in dorsal horn at both day 7 and day 14 following chemotherapy, but neuronal (connexin 36 [Cx36]) and oligodendrocyte (connexin 32 [Cx32]) gap junction proteins did not show any change. Blockade of astrocyte gap junction with carbenoxolone (CBX) prevented oxaliplatin-induced mechanical hypersensitivity in a dose-dependent manner and the increase of spinal GFAP expression, but had no effect once the mechanical hypersensitivity induced by oxaliplatin had fully developed. These results suggest that oxaliplatin chemotherapy induces the activation of spinal astrocytes and this is accompanied by increased expression of astrocyte-astrocyte gap junction connections via Cx43. These alterations in spinal astrocytes appear to contribute to the induction but not the maintenance of oxaliplatin-induced mechanical hypersensitivity. Combined, these results suggest that targeting spinal astrocyte/astrocyte-specific gap junction could be a new therapeutic strategy to prevent oxaliplatin-induced neuropathy. Spinal astrocytes but not microglia were recently shown to be recruited in paclitaxel-related chemoneuropathy. Here, spinal astrocyte gap junctions are shown to play an important role in the induction of oxaliplatin neuropathy. Copyright © 2013 American Pain Society. Published by Elsevier Inc. All rights reserved.

Evidence for Altered Ca2+ Handling in Growth Associated Protein 43-Knockout Skeletal Muscle

PubMed Central

Caprara, Giusy A.; Morabito, Caterina; Perni, Stefano; Navarra, Riccardo; Guarnieri, Simone; Mariggiò, Maria A.

2016-01-01

Neuronal growth-associated protein 43 (GAP43) has crucial roles in the nervous system, and during development, regeneration after injury, and learning and memory. GAP43 is expressed in mouse skeletal muscle fibers and satellite cells, with suggested its involvement in intracellular Ca2+ handling. However, the physiological role of GAP43 in muscle remains unknown. Using a GAP43-knockout (GAP43−/−) mouse, we have defined the role of GAP43 in skeletal muscle. GAP43−/− mice showed low survival beyond weaning, reduced adult body weight, decreased muscle strength, and changed myofiber ultrastructure, with no significant differences in the expression of markers of satellite cell and myotube progression through the myogenic program. Thus, GAP43 expression is involved in timing of muscle maturation in-vivo. Intracellular Ca2+ measurements in-vitro in myotubes revealed GAP43 involvement in Ca2+ handling. In the absence of GAP43 expression, the spontaneous Ca2+ variations had greater amplitudes and higher frequency. In GAP43−/− myotubes, also the intracellular Ca2+ variations induced by the activation of dihydropyridine and ryanodine Ca2+ channels, resulted modified. These evidences suggested dysregulation of Ca2+ homeostasis. The emerging hypothesis indicates that GAP43 interacts with calmodulin to indirectly modulate the activities of dihydropyridine and ryanodine Ca2+ channels. This thus influences intracellular Ca2+ dynamics and its related intracellular patterns, from functional excitation-contraction coupling, to cell metabolism, and gene expression. PMID:27833566

A Co-operative Regulation of Neuronal Excitability by UNC-7 Innexin and NCA/NALCN Leak Channel

PubMed Central

2011-01-01

Gap junctions mediate the electrical coupling and intercellular communication between neighboring cells. Some gap junction proteins, namely connexins and pannexins in vertebrates, and innexins in invertebrates, may also function as hemichannels. A conserved NCA/Dmα1U/NALCN family cation leak channel regulates the excitability and activity of vertebrate and invertebrate neurons. In the present study, we describe a genetic and functional interaction between the innexin UNC-7 and the cation leak channel NCA in Caenorhabditis elegans neurons. While the loss of the neuronal NCA channel function leads to a reduced evoked postsynaptic current at neuromuscular junctions, a simultaneous loss of the UNC-7 function restores the evoked response. The expression of UNC-7 in neurons reverts the effect of the unc-7 mutation; moreover, the expression of UNC-7 mutant proteins that are predicted to be unable to form gap junctions also reverts this effect, suggesting that UNC-7 innexin regulates neuronal activity, in part, through gap junction-independent functions. We propose that, in addition to gap junction-mediated functions, UNC-7 innexin may also form hemichannels to regulate C. elegans' neuronal activity cooperatively with the NCA family leak channels. PMID:21489288

Differential regulation of the Rac1 GTPase-activating protein (GAP) BCR during oxygen/glucose deprivation in hippocampal and cortical neurons.

PubMed

Smith, Katharine R; Rajgor, Dipen; Hanley, Jonathan G

2017-12-08

Brain ischemia causes oxygen and glucose deprivation (OGD) in neurons, triggering a cascade of events leading to synaptic accumulation of glutamate. Excessive activation of glutamate receptors causes excitotoxicity and delayed cell death in vulnerable neurons. Following global cerebral ischemia, hippocampal CA1 pyramidal neurons are more vulnerable to injury than their cortical counterparts, but the mechanisms that underlie this difference are unclear. Signaling via Rho-family small GTPases, their upstream guanine nucleotide exchange factors, and GTPase-activating proteins (GAPs) is differentially dysregulated in response to OGD/ischemia in hippocampal and cortical neurons. Increased Rac1 activity caused by OGD/ischemia contributes to neuronal death in hippocampal neurons via diverse effects on NADPH oxidase activity and dendritic spine morphology. The Rac1 guanine nucleotide exchange factor Tiam1 mediates an OGD-induced increase in Rac1 activity in hippocampal neurons; however, the identity of an antagonistic GAP remains elusive. Here we show that the Rac1 GAP breakpoint cluster region (BCR) associates with NMDA receptors (NMDARs) along with Tiam1 and that this protein complex is more abundant in hippocampal compared with cortical neurons. Although total BCR is similar in the two neuronal types, BCR is more active in hippocampal compared with cortical neurons. OGD causes an NMDAR- and Ca 2+ -permeable AMPAR-dependent deactivation of BCR in hippocampal but not cortical neurons. BCR knockdown occludes OGD-induced Rac1 activation in hippocampal neurons. Furthermore, disrupting the Tiam1-NMDAR interaction with a fragment of Tiam1 blocks OGD-induced Tiam1 activation but has no effect on the deactivation of BCR. This work identifies BCR as a critical player in Rac1 regulation during OGD in hippocampal neurons. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Interactions of Pannexin1 channels with purinergic and NMDA receptor channels.

PubMed

Li, Shuo; Bjelobaba, Ivana; Stojilkovic, Stanko S

2018-01-01

Pannexins are a three-member family of vertebrate plasma membrane spanning molecules that have homology to the invertebrate gap junction forming proteins, the innexins. However, pannexins do not form gap junctions but operate as plasma membrane channels. The best-characterized member of these proteins, Pannexin1 (Panx1) was suggested to be functionally associated with purinergic P2X and N-methyl-D-aspartate (NMDA) receptor channels. Activation of these receptor channels by their endogenous ligands leads to cross-activation of Panx1 channels. This in turn potentiates P2X and NMDA receptor channel signaling. Two potentiation concepts have been suggested: enhancement of the current responses and/or sustained receptor channel activation by ATP released through Panx1 pore and adenosine generated by ectonucleotidase-dependent dephosphorylation of ATP. Here we summarize the current knowledge and hypotheses about interactions of Panx1 channels with P2X and NMDA receptor channels. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve. Published by Elsevier B.V.

Stress Conditions Promote Yeast Gap1 Permease Ubiquitylation and Down-regulation via the Arrestin-like Bul and Aly Proteins*

PubMed Central

Crapeau, Myriam; Merhi, Ahmad; André, Bruno

2014-01-01

Gap1, the yeast general amino acid permease, is a convenient model for studying how the intracellular traffic of membrane transporters is regulated. Present at the plasma membrane under poor nitrogen supply conditions, it undergoes ubiquitylation, endocytosis, and degradation upon activation of the TORC1 kinase complex in response to an increase in internal amino acids. This down-regulation is stimulated by TORC1-dependent phosphoinhibition of the Npr1 kinase, resulting in activation by dephosphorylation of the arrestin-like Bul1 and Bul2 adaptors recruiting the Rsp5 ubiquitin ligase to Gap1. We report here that Gap1 is also down-regulated when cells are treated with the TORC1 inhibitor rapamycin or subjected to various stresses and that a lack of the Tco89 subunit of TORC1 causes constitutive Gap1 down-regulation. Both the Bul1 and Bul2 and the Aly1 and Aly2 arrestin-like adaptors of Rsp5 promote this down-regulation without undergoing dephosphorylation. Furthermore, they act via the C-terminal regions of Gap1 not involved in ubiquitylation in response to internal amino acids, whereas a Gap1 mutant altered in the N-terminal tail and resistant to ubiquitylation by internal amino acids is efficiently down-regulated under stress via the Bul and Aly adaptors. Although the Bul proteins mediate Gap1 ubiquitylation of two possible lysines, Lys-9 and Lys-16, the Aly proteins promote ubiquitylation of the Lys-16 residue only. This stress-induced pathway of Gap1 down-regulation targets other permeases as well, and it likely allows cells facing adverse conditions to retrieve amino acids from permease degradation. PMID:24942738

Modulation of Brain Hemichannels and Gap Junction Channels by Pro-Inflammatory Agents and Their Possible Role in Neurodegeneration

PubMed Central

Sáez, Pablo J.; Shoji, Kenji F.; Schalper, Kurt A.; Palacios–Prado, Nicolás; Velarde, Victoria; Giaume, Christian; Bennett, Michael V.L.; Sáez, Juan C.

2009-01-01

Abstract In normal brain, neurons, astrocytes, and oligodendrocytes, the most abundant and active cells express pannexins and connexins, protein subunits of two families forming membrane channels. Most available evidence indicates that in mammals endogenously expressed pannexins form only hemichannels and connexins form both gap junction channels and hemichannels. Whereas gap junction channels connect the cytoplasm of contacting cells and coordinate electric and metabolic activity, hemichannels communicate the intra- and extracellular compartments and serve as a diffusional pathway for ions and small molecules. A subthreshold stimulation by acute pathological threatening conditions (e.g., global ischemia subthreshold for cell death) enhances neuronal Cx36 and glial Cx43 hemichannel activity, favoring ATP release and generation of preconditioning. If the stimulus is sufficiently deleterious, microglia become overactivated and release bioactive molecules that increase the activity of hemichannels and reduce gap junctional communication in astroglial networks, depriving neurons of astrocytic protective functions, and further reducing neuronal viability. Continuous glial activation triggered by low levels of anomalous proteins expressed in several neurodegenerative diseases induce glial hemichannel and gap junction channel disorders similar to those of acute inflammatory responses triggered by ischemia or infectious diseases. These changes are likely to occur in diverse cell types of the CNS and contribute to neurodegeneration during inflammatory process. Antiox. Redox Signal. 11, 369–399. PMID:18816186

MRP transporters as membrane machinery in the bradykinin-inducible export of ATP.

PubMed

Zhao, Yumei; Migita, Keisuke; Sun, Jing; Katsuragi, Takeshi

2010-04-01

Adenosine triphosphate (ATP) plays the role of an autocrine/paracrine signal molecule in a variety of cells. So far, however, the membrane machinery in the export of intracellular ATP remains poorly understood. Activation of B2-receptor with bradykinin-induced massive release of ATP from cultured taenia coli smooth muscle cells. The evoked release of ATP was unaffected by gap junction hemichannel blockers, such as 18alpha-glycyrrhetinic acid and Gap 26. Furthermore, the cystic fibrosis transmembrane regulator (CFTR) coupled Cl(-) channel blockers, CFTR(inh)172, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, Gd3(+) and glibenclamide, failed to suppress the export of ATP by bradykinin. On the other, the evoked release of ATP was greatly reduced by multidrug resistance protein (MRP) transporter inhibitors, MK-571, indomethacin, and benzbromarone. From western blotting analysis, blots of MRP 1 protein only, but not MRP 2 and MRP 3 protein, appeared at 190 kD. However, the MRP 1 protein expression was not enhanced after loading with 1 muM bradykinin for 5 min. Likewise, niflumic acid and fulfenamic acid, Ca2(+)-activated Cl(-) channel blockers, largely abated the evoked release of ATP. The possibility that the MRP transporter system couples with Ca2(+)-activated Cl(-) channel activities is discussed here. These findings suggest that MRP transporters, probably MRP 1, unlike CFTR-Cl(-) channels and gap junction hemichannels, may contribute as membrane machinery to the export of ATP induced by G-protein-coupled receptor stimulation.

Ancient Complexity, Opisthokont Plasticity, and Discovery of the 11th Subfamily of Arf GAP Proteins

PubMed Central

Schlacht, Alexander; Mowbrey, Kevin; Elias, Marek; Kahn, Richard A.; Dacks, Joel B.

2013-01-01

The organelle paralogy hypothesis is one model for the acquisition of non-endosymbiotic organelles, generated from molecular evolutionary analyses of proteins encoding specificity in the membrane traffic system. GTPase Activating Proteins (GAPs) for the ADP-ribosylation factor (Arfs) GTPases are additional regulators of the kinetics and fidelity of membrane traffic. Here we describe molecular evolutionary analyses of Arf GAP protein family. Of the ten subfamilies previously defined in humans, we find that five were likely present in the Last Eukaryotic Common Ancestor (LECA). Of the three more recently derived subfamilies, one was likely present in the ancestor of opisthokonts (animals and fungi) and apusomonads (flagellates classified as the sister lineage to opisthokonts), while two arose in the holozoan lineage. We also propose to have identified a novel ancient subfamily (ArfGAPC2), present in diverse eukaryotes but which is lost frequently, including in the opisthokonts. Surprisingly few ancient domains accompanying the ArfGAP domain were identified, in marked contrast to the extensively decorated human Arf GAPs. Phylogenetic analyses of the subfamilies reveal patterns of single and multiple gene duplications specific to the Holozoa, to some degree mirroring evolution of Arf GAP targets, the Arfs. Conservation, and lack thereof, of various residues in the ArfGAP structure provide contextualization of previously identified functional amino acids and their application to Arf GAP biology in general. Overall, our results yield insights into current Arf GAP biology, reveal complexity in the ancient eukaryotic ancestor, and integrate the Arf GAP family into a proposed mechanism for the evolution of non-endosymbiotic organelles. PMID:23433073

Measurement of Single Channel Currents from Cardiac Gap Junctions

NASA Astrophysics Data System (ADS)

Veenstra, Richard D.; Dehaan, Robert L.

1986-08-01

Cardiac gap junctions consist of arrays of integral membrane proteins joined across the intercellular cleft at points of cell-to-cell contact. These junctional proteins are thought to form pores through which ions can diffuse from cytosol to cytosol. By monitoring whole-cell currents in pairs of embryonic heart cells with two independent patch-clamp circuits, the properties of single gap junction channels have been investigated. These channels had a conductance of about 165 picosiemens and underwent spontaneous openings and closings that were independent of voltage. Channel activity and macroscopic junctional conductance were both decreased by the uncoupling agent 1-octanol.

Identification of RhoGAP22 as an Akt-Dependent Regulator of Cell Motility in Response to Insulin▿‡

PubMed Central

Rowland, Alexander F.; Larance, Mark; Hughes, William E.; James, David E.

2011-01-01

Insulin exerts many of its metabolic actions via the canonical phosphatidylinositide 3 kinase (PI3K)/Akt pathway, leading to phosphorylation and 14-3-3 binding of key metabolic targets. We previously identified a GTPase-activating protein (GAP) for Rac1 called RhoGAP22 as an insulin-responsive 14-3-3 binding protein. Insulin increased 14-3-3 binding to RhoGAP22 fourfold, and this effect was PI3K dependent. We identified two insulin-responsive 14-3-3 binding sites (pSer16 and pSer395) within RhoGAP22, and mutagenesis studies revealed a complex interplay between the phosphorylation at these two sites. Mutating Ser16 to alanine blocked 14-3-3 binding to RhoGAP22 in vivo, and phosphorylation at Ser16 was mediated by the kinase Akt. Overexpression of a mutant RhoGAP22 that was unable to bind 14-3-3 reduced cell motility in NIH-3T3 fibroblasts, and this effect was dependent on a functional GAP domain. Mutation of the catalytic arginine of the GAP domain of RhoGAP22 potentiated growth factor-stimulated Rac1 GTP loading. We propose that insulin and possibly growth factors such as platelet-derived growth factor may play a novel role in regulating cell migration and motility via the Akt-dependent phosphorylation of RhoGAP22, leading to modulation of Rac1 activity. PMID:21969604

Hexadecameric structure of an invertebrate gap junction channel.

PubMed

Oshima, Atsunori; Matsuzawa, Tomohiro; Murata, Kazuyoshi; Tani, Kazutoshi; Fujiyoshi, Yoshinori

2016-03-27

Innexins are invertebrate-specific gap junction proteins with four transmembrane helices. These proteins oligomerize to constitute intercellular channels that allow for the passage of small signaling molecules associated with neural and muscular electrical activity. In contrast to the large number of structural and functional studies of connexin gap junction channels, few structural studies of recombinant innexin channels are reported. Here we show the three-dimensional structure of two-dimensionally crystallized Caenorhabditis elegans innexin-6 (INX-6) gap junction channels. The N-terminal deleted INX-6 proteins are crystallized in lipid bilayers. The three-dimensional reconstruction determined by cryo-electron crystallography reveals that a single INX-6 gap junction channel comprises 16 subunits, a hexadecamer, in contrast to chordate connexin channels, which comprise 12 subunits. The channel pore diameters at the cytoplasmic entrance and extracellular gap region are larger than those of connexin26. Two bulb densities are observed in each hemichannel, one in the pore and the other at the cytoplasmic side of the hemichannel in the channel pore pathway. These findings imply a structural diversity of gap junction channels among multicellular organisms. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Regulator of G-protein signalling and GoLoco proteins suppress TRPC4 channel function via acting at Gαi/o.

PubMed

Jeon, Jae-Pyo; Thakur, Dhananjay P; Tian, Jin-Bin; So, Insuk; Zhu, Michael X

2016-05-15

Transient receptor potential canonical 4 (TRPC4) forms non-selective cation channels implicated in the regulation of diverse physiological functions. Previously, TRPC4 was shown to be activated by the Gi/o subgroup of heterotrimeric G-proteins involving Gαi/o, rather than Gβγ, subunits. Because the lifetime and availability of Gα-GTP are regulated by regulators of G-protein signalling (RGS) and Gαi/o-Loco (GoLoco) domain-containing proteins via their GTPase-activating protein (GAP) and guanine-nucleotide-dissociation inhibitor (GDI) functions respectively, we tested how RGS and GoLoco domain proteins affect TRPC4 currents activated via Gi/o-coupled receptors. Using whole-cell patch-clamp recordings, we show that both RGS and GoLoco proteins [RGS4, RGS6, RGS12, RGS14, LGN or activator of G-protein signalling 3 (AGS3)] suppress receptor-mediated TRPC4 activation without causing detectable basal current or altering surface expression of the channel protein. The inhibitory effects are dependent on the GAP and GoLoco domains and facilitated by enhancing membrane targeting of the GoLoco protein AGS3. In addition, RGS, but not GoLoco, proteins accelerate desensitization of receptor-activation evoked TRPC4 currents. The inhibitory effects of RGS and GoLoco domains are additive and are most prominent with RGS12 and RGS14, which contain both RGS and GoLoco domains. Our data support the notion that the Gα, but not Gβγ, arm of the Gi/o signalling is involved in TRPC4 activation and unveil new roles for RGS and GoLoco domain proteins in fine-tuning TRPC4 activities. The versatile and diverse functions of RGS and GoLoco proteins in regulating G-protein signalling may underlie the complexity of receptor-operated TRPC4 activation in various cell types under different conditions. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Increased Epithelial Gap Density in the Noninflamed Duodenum of Children With Inflammatory Bowel Diseases.

PubMed

Zaidi, Deenaz; Bording-Jorgensen, Michael; Huynh, Hien Q; Carroll, Matthew W; Turcotte, Jean-Francois; Sergi, Consolato; Liu, Julia; Wine, Eytan

2016-12-01

Inflammatory bowel diseases (IBD) present commonly in childhood, with unknown etiology, but an important role for the epithelial lining is suggested. Epithelial cell extrusion, measured by counting gaps between epithelial cells, is higher in adult patients with Crohn disease (CD) than in controls. Our objectives were to compare epithelial gaps in the duodenum of IBD and non-IBD pediatric patients, to study the correlation between epithelial gaps, inflammation, and disease activity, and identify potential mechanisms. Epithelial gap density of the duodenum was evaluated using probe-based confocal laser endomicroscopy in 26 pediatric patients with IBD (16 CD, 10 ulcerative colitis [UC]) and 17 non-IBD controls during endoscopy. Epithelial gaps were correlated with serum inflammatory markers, disease activity indices, and intraepithelial lymphocytes. A panel of 10 inflammatory cytokines and expression of TNFAIP3 (A20; inhibits NF-κβ-induced inflammation) were analyzed in duodenal and ileal biopsies. Confocal imaging showed significantly higher epithelial gap density in patients with IBD, including UC. Interleukin (IL)-2 and IL-8 were higher in duodenal but not ileal biopsies of patients with UC. No significant correlation was present between C-reactive protein, erythrocyte sedimentation rate, disease activity indices, and epithelial gaps in patients with UC. In patients with CD, C-reactive protein positively correlated with epithelial gaps. A20 expression in the duodenum was unchanged among non-IBD and IBD cases. Duodenal epithelial gaps are increased in pediatric patients with IBD (including UC) but are unrelated to inflammation. This suggests that altered epithelial barrier is an important systemic feature of pediatric IBD and is not only secondary to inflammation.

Glyceraldehyde-3-phosphate dehydrogenase: a universal internal control for Western blots in prokaryotic and eukaryotic cells.

PubMed

Wu, Yonghong; Wu, Min; He, Guowei; Zhang, Xiao; Li, Weiguang; Gao, Yan; Li, Zhihui; Wang, Zhaoyan; Zhang, Chenggang

2012-04-01

In the current study, we examined the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in a number of organisms and the stability of GAPDH under various conditions. Our results revealed that GAPDH is present in multiple Escherichia coli strains, the yeast strain GS115, Caenorhabditis elegans, rat PC12 cells, and both mouse and rat brain. Furthermore, GAPDH was stably expressed under different concentrations of inducer and at different times of induction in E. coli (BL21) cells and yeast GS115 cells. Stable expression of GAPDH protein was also observed in C.elegans and PC12 cells that were treated with different concentrations of paraquat or sodium sulfite, respectively. In addition, we were able to detect and identify the endogenous gapA protein in E.coli via immunoprecipitation and MALDI-TOF-MS analysis. Endogenous gapA protein and exogenously expressed (subcloned) GAPDH proteins were detected in E. coli BL21 but not for gapC. With the exception of gapC in E. coli, the various isoforms of GAPDH possessed enzymatic activity. Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

EPI64B Acts as a GTPase-activating Protein for Rab27B in Pancreatic Acinar Cells*

PubMed Central

Hou, Yanan; Chen, Xuequn; Tolmachova, Tatyana; Ernst, Stephen A.; Williams, John A.

2013-01-01

The small GTPase Rab27B localizes to the zymogen granule membranes and plays an important role in regulating protein secretion by pancreatic acinar cells, as does Rab3D. A common guanine nucleotide exchange factor (GEF) for Rab3 and Rab27 has been reported; however, the GTPase-activating protein (GAP) specific for Rab27B has not been identified. In this study, the expression in mouse pancreatic acini of two candidate Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins, EPI64 (TBC1D10A) and EPI64B (TBC1D10B), was first demonstrated. Their GAP activity on digestive enzyme secretion was examined by adenovirus-mediated overexpression of EPI64 and EPI64B in isolated pancreatic acini. EPI64B almost completely abolished the GTP-bound form of Rab27B, without affecting GTP-Rab3D. Overexpression of EPI64B also enhanced amylase release. This enhanced release was independent of Rab27A, but dependent on Rab27B, as shown using acini from genetically modified mice. EPI64 had a mild effect on both GTP-Rab27B and amylase release. Co-overexpression of EPI64B with Rab27B can reverse the inhibitory effect of Rab27B on amylase release. Mutations that block the GAP activity decreased the inhibitory effect of EPI64B on the GTP-bound state of Rab27B and abolished the enhancing effect of EPI64B on the amylase release. These data suggest that EPI64B can serve as a potential physiological GAP for Rab27B and thereby participate in the regulation of exocytosis in pancreatic acinar cells. PMID:23671284

Reconstitution of the Recombinant RanBP2 SUMO E3 Ligase Complex.

PubMed

Ritterhoff, Tobias; Das, Hrishikesh; Hao, Yuqing; Sakin, Volkan; Flotho, Annette; Werner, Andreas; Melchior, Frauke

2016-01-01

One of the few proteins that have SUMO E3 ligase activity is the 358 kDa nucleoporin RanBP2 (Nup358). While small fragments of RanBP2 can stimulate SUMOylation in vitro, the physiologically relevant E3 ligase is a stable multi-subunit complex comprised of RanBP2, SUMOylated RanGAP1, and Ubc9. Here, we provide a detailed protocol to in vitro reconstitute the RanBP2 SUMO E3 ligase complex. With the exception of RanBP2, reconstitution involves untagged full-length proteins. We describe the bacterial expression and purification of all complex components, namely an 86 kDa His-tagged RanBP2 fragment, the SUMO E2-conjugating enzyme Ubc9, RanGAP1, and SUMO1, and we provide a protocol for quantitative SUMOylation of RanGAP1. Finally, we present details for the assembly and final purification of the catalytically active RanBP2/RanGAP1*SUMO1/Ubc9 complex.

Structure and Function of the Intracellular Region of the Plexin-B1 Transmembrane Receptor*

PubMed Central

Tong, Yufeng; Hota, Prasanta K.; Penachioni, Junia Y.; Hamaneh, Mehdi B.; Kim, SoonJeung; Alviani, Rebecca S.; Shen, Limin; He, Hao; Tempel, Wolfram; Tamagnone, Luca; Park, Hee-Won; Buck, Matthias

2009-01-01

Members of the plexin family are unique transmembrane receptors in that they interact directly with Rho family small GTPases; moreover, they contain a GTPase-activating protein (GAP) domain for R-Ras, which is crucial for plexin-mediated regulation of cell motility. However, the functional role and structural basis of the interactions between the different intracellular domains of plexins remained unclear. Here we present the 2.4 Å crystal structure of the complete intracellular region of human plexin-B1. The structure is monomeric and reveals that the GAP domain is folded into one structure from two segments, separated by the Rho GTPase binding domain (RBD). The RBD is not dimerized, as observed previously. Instead, binding of a conserved loop region appears to compete with dimerization and anchors the RBD to the GAP domain. Cell-based assays on mutant proteins confirm the functional importance of this coupling loop. Molecular modeling based on structural homology to p120GAP·H-Ras suggests that Ras GTPases can bind to the plexin GAP region. Experimentally, we show that the monomeric intracellular plexin-B1 binds R-Ras but not H-Ras. These findings suggest that the monomeric form of the intracellular region is primed for GAP activity and extend a model for plexin activation. PMID:19843518

Cross-Species Analyses Identify the BNIP-2 and Cdc42GAP Homology (BCH) Domain as a Distinct Functional Subclass of the CRAL_TRIO/Sec14 Superfamily

PubMed Central

Gupta, Anjali Bansal; Wee, Liang En; Zhou, Yi Ting; Hortsch, Michael; Low, Boon Chuan

2012-01-01

The CRAL_TRIO protein domain, which is unique to the Sec14 protein superfamily, binds to a diverse set of small lipophilic ligands. Similar domains are found in a range of different proteins including neurofibromatosis type-1, a Ras GTPase-activating Protein (RasGAP) and Rho guanine nucleotide exchange factors (RhoGEFs). Proteins containing this structural protein domain exhibit a low sequence similarity and ligand specificity while maintaining an overall characteristic three-dimensional structure. We have previously demonstrated that the BNIP-2 and Cdc42GAP Homology (BCH) protein domain, which shares a low sequence homology with the CRAL_TRIO domain, can serve as a regulatory scaffold that binds to Rho, RhoGEFs and RhoGAPs to control various cell signalling processes. In this work, we investigate 175 BCH domain-containing proteins from a wide range of different organisms. A phylogenetic analysis with ∼100 CRAL_TRIO and similar domains from eight representative species indicates a clear distinction of BCH-containing proteins as a novel subclass within the CRAL_TRIO/Sec14 superfamily. BCH-containing proteins contain a hallmark sequence motif R(R/K)h(R/K)(R/K)NL(R/K)xhhhhHPs (‘h’ is large and hydrophobic residue and ‘s’ is small and weekly polar residue) and can be further subdivided into three unique subtypes associated with BNIP-2-N, macro- and RhoGAP-type protein domains. A previously unknown group of genes encoding ‘BCH-only’ domains is also identified in plants and arthropod species. Based on an analysis of their gene-structure and their protein domain context we hypothesize that BCH domain-containing genes evolved through gene duplication, intron insertions and domain swapping events. Furthermore, we explore the point of divergence between BCH and CRAL-TRIO proteins in relation to their ability to bind small GTPases, GAPs and GEFs and lipid ligands. Our study suggests a need for a more extensive analysis of previously uncharacterized BCH, ‘BCH-like’ and CRAL_TRIO-containing proteins and their significance in regulating signaling events involving small GTPases. PMID:22479462

The Arf6 GTPase-activating proteins ARAP2 and ACAP1 define distinct endosomal compartments that regulate integrin α5β1 traffic.

PubMed

Chen, Pei-Wen; Luo, Ruibai; Jian, Xiaoying; Randazzo, Paul A

2014-10-31

Arf6 and the Arf6 GTPase-activating protein (GAP) ACAP1 are established regulators of integrin traffic important to cell adhesion and migration. However, the function of Arf6 with ACAP1 cannot explain the range of Arf6 effects on integrin-based structures. We propose that Arf6 has different functions determined, in part, by the associated Arf GAP. We tested this idea by comparing the Arf6 GAPs ARAP2 and ACAP1. We found that ARAP2 and ACAP1 had opposing effects on apparent integrin β1 internalization. ARAP2 knockdown slowed, whereas ACAP1 knockdown accelerated, integrin β1 internalization. Integrin β1 association with adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif (APPL)-positive endosomes and EEA1-positive endosomes was affected by ARAP2 knockdown and depended on ARAP2 GAP activity. ARAP2 formed a complex with APPL1 and colocalized with Arf6 and APPL in a compartment distinct from the Arf6/ACAP1 tubular recycling endosome. In addition, although ACAP1 and ARAP2 each colocalized with Arf6, they did not colocalize with each other and had opposing effects on focal adhesions (FAs). ARAP2 overexpression promoted large FAs, but ACAP1 overexpression reduced FAs. Taken together, the data support a model in which Arf6 has at least two sites of opposing action defined by distinct Arf6 GAPs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

ACBD3 functions as a scaffold to organize the Golgi stacking proteins and a Rab33b-GAP.

PubMed

Yue, Xihua; Bao, Mengjing; Christiano, Romain; Li, Siyang; Mei, Jia; Zhu, Lianhui; Mao, Feifei; Yue, Qiang; Zhang, Panpan; Jing, Shuaiyang; Rothman, James E; Qian, Yi; Lee, Intaek

2017-09-01

Golgin45 plays important roles in Golgi stack assembly and is known to bind both the Golgi stacking protein GRASP55 and Rab2 in the medial-Golgi cisternae. In this study, we sought to further characterize the cisternal adhesion complex using a proteomics approach. We report here that Acyl-CoA binding domain containing 3 (ACBD3) is likely to be a novel binding partner of Golgin45. ACBD3 interacts with Golgin45 via its GOLD domain, while its co-expression significantly increases Golgin45 targeting to the Golgi. Furthermore, ACBD3 recruits TBC1D22, a Rab33b GTPase activating protein (GAP), to a large multi-protein complex containing Golgin45 and GRASP55. These results suggest that ACBD3 may provide a scaffolding to organize the Golgi stacking proteins and a Rab33b-GAP at the medial-Golgi. © 2017 Federation of European Biochemical Societies.

Astroglial Metabolic Networks Sustain Hippocampal Synaptic Transmission

NASA Astrophysics Data System (ADS)

Rouach, Nathalie; Koulakoff, Annette; Abudara, Veronica; Willecke, Klaus; Giaume, Christian

2008-12-01

Astrocytes provide metabolic substrates to neurons in an activity-dependent manner. However, the molecular mechanisms involved in this function, as well as its role in synaptic transmission, remain unclear. Here, we show that the gap-junction subunit proteins connexin 43 and 30 allow intercellular trafficking of glucose and its metabolites through astroglial networks. This trafficking is regulated by glutamatergic synaptic activity mediated by AMPA receptors. In the absence of extracellular glucose, the delivery of glucose or lactate to astrocytes sustains glutamatergic synaptic transmission and epileptiform activity only when they are connected by gap junctions. These results indicate that astroglial gap junctions provide an activity-dependent intercellular pathway for the delivery of energetic metabolites from blood vessels to distal neurons.

Astroglial metabolic networks sustain hippocampal synaptic transmission.

PubMed

Rouach, Nathalie; Koulakoff, Annette; Abudara, Veronica; Willecke, Klaus; Giaume, Christian

2008-12-05

Astrocytes provide metabolic substrates to neurons in an activity-dependent manner. However, the molecular mechanisms involved in this function, as well as its role in synaptic transmission, remain unclear. Here, we show that the gap-junction subunit proteins connexin 43 and 30 allow intercellular trafficking of glucose and its metabolites through astroglial networks. This trafficking is regulated by glutamatergic synaptic activity mediated by AMPA receptors. In the absence of extracellular glucose, the delivery of glucose or lactate to astrocytes sustains glutamatergic synaptic transmission and epileptiform activity only when they are connected by gap junctions. These results indicate that astroglial gap junctions provide an activity-dependent intercellular pathway for the delivery of energetic metabolites from blood vessels to distal neurons.

Wnt-related SynGAP1 is a neuroprotective factor of glutamatergic synapses against Aβ oligomers

PubMed Central

Codocedo, Juan F.; Montecinos-Oliva, Carla; Inestrosa, Nibaldo C.

2015-01-01

Wnt-5a is a synaptogenic factor that modulates glutamatergic synapses and generates neuroprotection against Aβ oligomers. It is known that Wnt-5a plays a key role in the adult nervous system and synaptic plasticity. Emerging evidence indicates that miRNAs are actively involved in the regulation of synaptic plasticity. Recently, we showed that Wnt-5a is able to control the expression of several miRNAs including miR-101b, which has been extensively studied in carcinogenesis. However, its role in brain is just beginning to be explored. That is why we aim to study the relationship between Wnt-5a and miRNAs in glutamatergic synapses. We performed in silico analysis which predicted that miR-101b may inhibit the expression of synaptic GTPase-Activating Protein (SynGAP1), a Ras GTPase-activating protein critical for the development of cognition and proper synaptic function. Through overexpression of miR-101b, we showed that miR-101b is able to regulate the expression of SynGAP1 in an hippocampal cell line. Moreover and consistent with a decrease of miR-101b, Wnt-5a enhances SynGAP expression in cultured hippocampal neurons. Additionally, Wnt-5a increases the activity of SynGAP in a time-dependent manner, with a similar kinetic to CaMKII phosphorylation. This also, correlates with a modulation in the SynGAP clusters density. On the other hand, Aβ oligomers permanently decrease the number of SynGAP clusters. Interestingly, when neurons are co-incubated with Wnt-5a and Aβ oligomers, we do not observe the detrimental effect of Aβ oligomers, indicating that, Wnt-5a protects neurons from the synaptic failure triggered by Aβ oligomers. Overall, our findings suggest that SynGAP1 is part of the signaling pathways induced by Wnt-5a. Therefore, possibility exists that SynGAP is involved in the synaptic protection against Aβ oligomers. PMID:26124704

Connexin43 synthesis, phosphorylation, and degradation in regulation of transient inhibition of gap junction intercellular communication by the phorbol ester TPA in rat liver epithelial cells.

PubMed

Rivedal, Edgar; Leithe, Edward

2005-01-15

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces transient inhibition of gap junction intercellular communication (GJIC) in several cell types. The initial block in GJIC has been attributed to protein kinase C (PKC) mediated phosphorylation of connexin gap junction proteins, including connexin43 (Cx43). Restoration of GJIC, associated with normalization of the Cx43 phosphorylation status, has been ascribed to different events, including dephosphorylation of Cx43 and de novo synthesis of Cx43 or other, non-gap junctional, proteins. The data presented suggest that restoration of GJIC during continuous TPA exposure in normal and transformed rat liver epithelial cells is dependent on synthesis of Cx43 protein, as well as the transport of already synthesized Cx43 from intracellular pools to the plasma membrane. Reactivation of inactivated Cx43 by dephosphorylation does not appear to be involved in the recovery of GJIC. Both PKC and MAP kinase is involved in TPA-induced degradation of Cx43 and inhibition of GJIC. We show that coincubation of TPA with the protein synthesis inhibitor cycloheximide or the transcription inhibitor actinomycin D results in synergistic enhancement of the level of activated ERK1/2. Together, the present data highlight Cx43 degradation and synthesis as critical determinants in TPA-induced modifications of cell-cell communication via gap junctions.

Molecular characterization of a novel RhoGAP, RRC-1 of the nematode Caenorhabditis elegans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Delawary, Mina; Nakazawa, Takanobu; Tezuka, Tohru

2007-06-01

The GTPase-activating proteins for Rho family GTPases (RhoGAP) transduce diverse intracellular signals by negatively regulating Rho family GTPase-mediated pathways. In this study, we have cloned and characterized a novel RhoGAP for Rac1 and Cdc42, termed RRC-1, from Caenorhabditis elegans. RRC-1 was highly homologous to mammalian p250GAP and promoted GTP hydrolysis of Rac1 and Cdc42 in cells. The rrc-1 mRNA was expressed in all life stages. Using an RRC-1::GFP fusion protein, we found that RRC-1 was localized to the coelomocytes, excretory cell, GLR cells, and uterine-seam cell in adult worms. These data contribute toward understanding the roles of Rho family GTPasesmore » in C. elegans.« less

Inhibition of gap junction currents by the abused solvent toluene.

PubMed

Del Re, Angelo M; Woodward, John J

2005-05-09

Abused inhalants are a large class of compounds that are inhaled for their intoxicating and mood altering effects. They include chemicals with known therapeutic uses such as anesthetic gases as well as volatile organic solvents like toluene that are found in paint thinners and adhesives. Because of their widespread commercial use and availability, inhalants are often among the first drugs that children encounter and use of these compounds is often associated with adverse acute and long-term consequences. The cellular and molecular sites of action for abused inhalants is not well known although recent studies report that toluene and other organic solvents alter the activity of specific ligand- and voltage-gated ion channels that regulate cellular excitability. As part of an ongoing effort to define molecular sites of action for abused inhalants, this study examined the effect of toluene on the function of gap junction proteins endogenously expressed in human embryonic kidney (HEK 293) cells. Gap junctions allow cell-to-cell electrical communication as well as passage of small molecular weight substances and are critical for synchronizing cellular activity in certain tissues. Gap junction currents in HEK 293 cells were measured during brief voltage steps using patch-clamp electrophysiology and were blocked by known gap junction blockers confirming expression of connexin proteins in these cells. Toluene dose-dependently inhibited these conductances with threshold effects appearing at approximately 0.4 mM and near complete inhibition occurring at concentrations of 1 mM and higher. The estimated EC50 value for toluene inhibition of gap junction currents in HEK 293 cells was 0.57 mM. The results of these studies suggest that volatile solvents including toluene may produce some of their effects by disrupting inter-cellular communication mediated by gap junction proteins.

Activation of ERK1/2 and PI3K/Akt by IGF-1 on GAP-43 expression in DRG neurons with excitotoxicity induced by glutamate in vitro.

PubMed

Liu, Zhen; Cai, Heng; Zhang, Ping; Li, Hao; Liu, Huaxiang; Li, Zhenzhong

2012-03-01

Insulin-like growth factor-1 (IGF-1) is a neurotrophic factor and plays an important role in promoting axonal growth from dorsal root ganglion (DRG) neurons. Whether IGF-1 influences growth-associated protein 43 (GAP-43) expression and activates the extracellular signal-regulated protein kinase (ERK1/2) and the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways in DRG neurons with excitotoxicity induced by glutamate (Glu) remains unknown. In this study, embryonic 15-day-old rat DRG explants were cultured for 48 h and then exposed to IGF-1, Glu, Glu + IGF-1, Glu + IGF-1 + PD98059, Glu + IGF-1 + LY294002, Glu + IGF-1 + PD98059 + LY294002 for additional 12 h. The DRG explants were continuously exposed to growth media as control. The levels of GAP-43 mRNA were detected by real time-PCR analysis. The protein levels of GAP-43, phosphorylated ERK1/2, phosphorylated Akt, total ERK1/2, and total Akt were detected by Western blot assay. GAP-43 expression in situ was determined by immunofluorescent labeling. Apoptotic cell death was monitored by Hoechst 33342 staining. IGF-1 alone increased GAP-43 and its mRNA levels in the absence of Glu. The decreased GAP-43 and its mRNA levels caused by Glu could be partially reversed by the presence of IGF-1. IGF-1 rescued neuronal cell death caused by Glu. Neither the ERK1/2 inhibitor PD98059 nor the PI3K inhibitor LY294002 blocked the effect of IGF-1, but both inhibitors together were effective. To validate the impact of GAP-43 expression by IGF-1, GAP-43 induction was blocked by administration of dexamethasone (DEX). IGF-1 partially rescued the decrease of GAP-43 and its mRNA levels induced by DEX. DEX induced an increase of cell apoptosis. IGF-1 may play an important role in neuroprotective effects on DRG neurons through regulating GAP-43 expression with excitotoxicity induced by Glu and the process was involved in both ERK1/2 and PI3K/Akt signaling pathways.

Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca2+ influx through cyclic nucleotide‐gated channels

PubMed Central

Bader, Almke; Bintig, Willem; Begandt, Daniela; Klett, Anne; Siller, Ina G.; Gregor, Carola; Schaarschmidt, Frank; Weksler, Babette; Romero, Ignacio; Couraud, Pierre‐Olivier; Hell, Stefan W.

2017-01-01

Key points Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell‐to‐cell diffusion of ions, metabolites and second messengers.Stimulation of the adenosine receptor subtype A2B increases the gap junction coupling in the human blood–brain barrier endothelial cell line hCMEC/D3.Although the increased gap junction coupling is cAMP‐dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase.We found that cAMP activates cyclic nucleotide‐gated (CNG) channels and thereby induces a Ca2+ influx, which leads to the increase in gap junction coupling.The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood–brain barrier. Abstract The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood–brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT‐PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 2‐phenylaminoadenosine (2‐PAA) on the gap junction coupling. We found that 2‐PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration‐dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2‐PAA‐related enhancement of gap junction coupling. In contrast, the cyclic nucleotide‐gated (CNG) channel inhibitor l‐cis‐diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+, suppressed the 2‐PAA‐related enhancement of gap junction coupling. Moreover, we observed a 2‐PAA‐dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP‐dependent manner. Ca2+ in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor‐dependent signalling of endothelial cells of the blood–brain barrier. PMID:28075020

Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca2+ influx through cyclic nucleotide-gated channels.

PubMed

Bader, Almke; Bintig, Willem; Begandt, Daniela; Klett, Anne; Siller, Ina G; Gregor, Carola; Schaarschmidt, Frank; Weksler, Babette; Romero, Ignacio; Couraud, Pierre-Olivier; Hell, Stefan W; Ngezahayo, Anaclet

2017-04-15

Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell-to-cell diffusion of ions, metabolites and second messengers. Stimulation of the adenosine receptor subtype A 2B increases the gap junction coupling in the human blood-brain barrier endothelial cell line hCMEC/D3. Although the increased gap junction coupling is cAMP-dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase. We found that cAMP activates cyclic nucleotide-gated (CNG) channels and thereby induces a Ca 2+ influx, which leads to the increase in gap junction coupling. The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood-brain barrier. The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A 2A and A 2B adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the gap junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of gap junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca 2+ with BAPTA, or the absence of external Ca 2+ , suppressed the 2-PAA-related enhancement of gap junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca 2+ influx by opening CNG channels in a cAMP-dependent manner. Ca 2+ in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood-brain barrier. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Molecular Mechanotransduction: how forces trigger cytoskeletal dynamics

NASA Astrophysics Data System (ADS)

Ehrlicher, Allen

2012-02-01

Mechanical stresses elicit cellular reactions mediated by chemical signals. Defective responses to forces underlie human medical disorders, such as cardiac failure and pulmonary injury. Despite detailed knowledge of the cytoskeleton's structure, the specific molecular switches that convert mechanical stimuli into chemical signals have remained elusive. Here we identify the actin-binding protein, filamin A (FLNa) as a central mechanotransduction element of the cytoskeleton by using Fluorescence Loss After photoConversion (FLAC), a novel high-speed alternative to FRAP. We reconstituted a minimal system consisting of actin filaments, FLNa and two FLNa-binding partners: the cytoplasmic tail of ß-integrin, and FilGAP. Integrins form an essential mechanical linkage between extracellular and intracellular environments, with ß integrin tails connecting to the actin cytoskeleton by binding directly to filamin. FilGAP is a FLNa-binding GTPase-activating protein specific for Rac, which in vivo regulates cell spreading and bleb formation. We demonstrate that both externally-imposed bulk shear and myosin II driven forces differentially regulate the binding of integrin and FilGAP to FLNa. Consistent with structural predictions, strain increases ß-integrin binding to FLNa, whereas it causes FilGAP to dissociate from FLNa, providing a direct and specific molecular basis for cellular mechanotransduction. These results identify the first molecular mechanotransduction element within the actin cytoskeleton, revealing that mechanical strain of key proteins regulates the binding of signaling molecules. Moreover, GAP activity has been shown to switch cell movement from mesenchymal to amoeboid motility, suggesting that mechanical forces directly impact the invasiveness of cancer.

SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting

PubMed Central

Siu, Fai Y.; Spanggord, Richard J.; Doudna, Jennifer A.

2007-01-01

The signal recognition particle (SRP) cotranslationally targets proteins to cell membranes by coordinated binding and release of ribosome-associated nascent polypeptides and a membrane-associated SRP receptor. GTP uptake and hydrolysis by the SRP-receptor complex govern this targeting cycle. Because no GTPase-activating proteins (GAPs) are known for the SRP and SRP receptor GTPases, however, it has been unclear whether and how GTP hydrolysis is stimulated during protein trafficking in vivo. Using both biochemical and genetic experiments, we show here that SRP RNA enhances GTPase activity of the SRP–receptor complex above a critical threshold required for cell viability. Furthermore, this stimulation is a property of the SRP RNA tetraloop. SRP RNA tetraloop mutants that confer defective growth phenotypes can assemble into SRP–receptor complexes, but fail to stimulate GTP hydrolysis in these complexes in vitro. Tethered hydroxyl radical probing data reveal that specific positioning of the RNA tetraloop within the SRP–receptor complex is required to stimulate GTPase activity to a level sufficient to support cell growth. These results explain why no external GAP is needed and why the phylogenetically conserved SRP RNA tetraloop is required in vivo. PMID:17164479

Gating connexin 43 channels reconstituted in lipid vesicles by mitogen-activated protein kinase phosphorylation.

PubMed

Kim, D Y; Kam, Y; Koo, S K; Joe, C O

1999-02-26

The regulation of gap junctional permeability by phosphorylation was examined in a model system in which connexin 43 (Cx43) gap junction hemichannels were reconstituted in lipid vesicles. Cx43 was immunoaffinity-purified from rat brain, and Cx43 channels were reconstituted into unilamellar phospholipid liposomes. The activities of the reconstituted channels were measured by monitoring liposome permeability. Liposomes containing the Cx43 protein were fractionated on the basis of permeability to sucrose using sedimentation in an iso-osmolar density gradient. The gradient allowed separation of the sucrose-permeable and -impermeable liposomes. Liposomes that were permeable to sucrose were also permeable to the communicating dye molecule lucifer yellow. Permeability, and therefore activity of the reconstituted Cx43 channels, were directly dependent on the state of Cx43 phosphorylation. The permeability of liposomes containing Cx43 channels was increased by treatment of liposomes with calf intestinal phosphatase. Moreover, liposomes formed with Cx43 that had been dephosphorylated by calf intestinal phosphatase treatment showed increased permeability to sucrose. The role of phosphorylation in the gating mechanism of Cx43 channels was supported further by the observation that phosphorylation of Cx43 by mitogen-activated protein kinase reversibly reduced the permeability of liposomes containing dephosphorylated Cx43. Our results show a direct correlation between gap junctional permeability and the phosphorylation state of Cx43.

An Arf-GAP promotes endocytosis and hyphal growth of Ashbya gossypii.

PubMed

Oscarsson, Therese; Walther, Andrea; Lengeler, Klaus B; Wendland, Jürgen

2017-12-29

The ADP-ribosylation factor (ARF) family of GTPases are highly conserved from yeast to human and regulate vesicle budding. Sec7 domain containing proteins stimulate the guanine nucleotide exchange on Arf proteins, while ARF-GTPase activating proteins stimulate the hydrolysis of GTP. Since vesicle trafficking is important for hyphal growth, we studied the Ashbya gossypii homolog of Saccharomyces cerevisiae ARF3 along with its putative GEF and GTPase-activating protein (GAP) encoded by YEL1 and GTS1, respectively. Deletion of YEL1 had no discernible phenotype and deletion of ARF3 had only a minor defect in vacuolar fusion. In contrast, deletion of GTS1 severely impaired hyphal growth, and mutants showed defects in the maintenance of polarity and the localization of cortical actin patches. The uptake of the lipophilic dye FM4-64 was delayed in gts1 hyphae, indicating a defect in endocytosis. Gts1 has several protein domains, of which the Arf-GAP domain is required for complementation of the gts1 mutant phenotype. GFP-tagged GTS1 under control of its endogenous promoter localized to the plasma membrane but was enriched at hyphal tips and septal sites corresponding to a role in polarized vesicle trafficking. Our results indicate that this ARF-GTPase module plays an important role for filamentous hyphal growth. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The E3 ubiquitin ligase NEDD4 induces endocytosis and lysosomal sorting of connexin 43 to promote loss of gap junctions.

PubMed

Totland, Max Z; Bergsland, Christian H; Fykerud, Tone A; Knudsen, Lars M; Rasmussen, Nikoline L; Eide, Peter W; Yohannes, Zeremariam; Sørensen, Vigdis; Brech, Andreas; Lothe, Ragnhild A; Leithe, Edward

2017-09-01

Intercellular communication via gap junctions has an important role in controlling cell growth and in maintaining tissue homeostasis. Connexin 43 (Cx43; also known as GJA1) is the most abundantly expressed gap junction channel protein in humans and acts as a tumor suppressor in multiple tissue types. Cx43 is often dysregulated at the post-translational level during cancer development, resulting in loss of gap junctions. However, the molecular basis underlying the aberrant regulation of Cx43 in cancer cells has remained elusive. Here, we demonstrate that the oncogenic E3 ubiquitin ligase NEDD4 regulates the Cx43 protein level in HeLa cells, both under basal conditions and in response to protein kinase C activation. Furthermore, overexpression of NEDD4, but not a catalytically inactive form of NEDD4, was found to result in nearly complete loss of gap junctions and increased lysosomal degradation of Cx43 in both HeLa and C33A cervical carcinoma cells. Collectively, the data provide new insights into the molecular basis underlying the regulation of gap junction size and represent the first evidence that an oncogenic E3 ubiquitin ligase promotes loss of gap junctions and Cx43 degradation in human carcinoma cells. © 2017. Published by The Company of Biologists Ltd.

Synaptic activity induces input-specific rearrangements in a targeted synaptic protein interaction network.

PubMed

Lautz, Jonathan D; Brown, Emily A; VanSchoiack, Alison A Williams; Smith, Stephen E P

2018-05-27

Cells utilize dynamic, network level rearrangements in highly interconnected protein interaction networks to transmit and integrate information from distinct signaling inputs. Despite the importance of protein interaction network dynamics, the organizational logic underlying information flow through these networks is not well understood. Previously, we developed the quantitative multiplex co-immunoprecipitation platform, which allows for the simultaneous and quantitative measurement of the amount of co-association between large numbers of proteins in shared complexes. Here, we adapt quantitative multiplex co-immunoprecipitation to define the activity dependent dynamics of an 18-member protein interaction network in order to better understand the underlying principles governing glutamatergic signal transduction. We first establish that immunoprecipitation detected by flow cytometry can detect activity dependent changes in two known protein-protein interactions (Homer1-mGluR5 and PSD-95-SynGAP). We next demonstrate that neuronal stimulation elicits a coordinated change in our targeted protein interaction network, characterized by the initial dissociation of Homer1 and SynGAP-containing complexes followed by increased associations among glutamate receptors and PSD-95. Finally, we show that stimulation of distinct glutamate receptor types results in different modular sets of protein interaction network rearrangements, and that cells activate both modules in order to integrate complex inputs. This analysis demonstrates that cells respond to distinct types of glutamatergic input by modulating different combinations of protein co-associations among a targeted network of proteins. Our data support a model of synaptic plasticity in which synaptic stimulation elicits dissociation of preexisting multiprotein complexes, opening binding slots in scaffold proteins and allowing for the recruitment of additional glutamatergic receptors. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Systematic mutational analysis of the intracellular regions of yeast Gap1 permease.

PubMed

Merhi, Ahmad; Gérard, Nicolas; Lauwers, Elsa; Prévost, Martine; André, Bruno

2011-04-19

The yeast general amino acid permease Gap1 is a convenient model for studying the intracellular trafficking of membrane proteins. Present at the plasma membrane when the nitrogen source is poor, it undergoes ubiquitin-dependent endocytosis and degradation upon addition of a good nitrogen source, e.g., ammonium. It comprises 12 transmembrane domains (TM) flanked by cytosol-facing N- and C-terminal tails (NT, CT). The NT of Gap1 contains the acceptor lysines for ubiquitylation and its CT includes a sequence essential to exit from the endoplasmic reticulum (ER). We used alanine-scanning mutagenesis to isolate 64 mutant Gap1 proteins altered in the NT, the CT, or one of the five TM-connecting intracellular loops (L2, -4, -6, -8 and -10). We found 17 mutations (in L2, L8, L10 and CT) impairing Gap1 exit from the ER. Of the 47 mutant proteins reaching the plasma membrane normally, two are unstable and rapidly down-regulated even when the nitrogen source is poor. Six others are totally inactive and another four, altered in a 16-amino-acid sequence in the NT, are resistant to ammonium-induced down-regulation. Finally, a mutation in L6 causes missorting of Gap1 from the secretory pathway to the vacuole. Interestingly, this direct vacuolar sorting seems to be independent of Gap1 ubiquitylation. This study illustrates the importance of multiple intracellular regions of Gap1 in its secretion, transport activity, and down-regulation.

Defect in the GTPase activating protein (GAP) function of eIF5 causes repression of GCN4 translation.

PubMed

Antony A, Charles; Alone, Pankaj V

2017-05-13

In eukaryotes, the eIF5 protein plays an important role in translation start site selection by providing the GAP (GTPase activating protein) function. However, in yeast translation initiation fidelity defective eIF5 G31R mutant causes preferential utilization of UUG as initiation codon and is termed as Suppressor of initiation codon (Sui - ) phenotype due to its hyper GTPase activity. The eIF5 G31R mutant dominantly represses GCN4 expression and confers sensitivity to 3-Amino-1,2,4-Trizole (3AT) induced starvation. The down-regulation of the GCN4 expression (Gcn - phenotype) in the eIF5 G31R mutant was not because of leaky scanning defects; rather was due to the utilization of upUUG initiation codons at the 5' regulatory region present between uORF1 and the main GCN4 ORF. Copyright © 2017 Elsevier Inc. All rights reserved.

RCP-driven α5β1 recycling suppresses Rac and promotes RhoA activity via the RacGAP1-IQGAP1 complex.

PubMed

Jacquemet, Guillaume; Green, David M; Bridgewater, Rebecca E; von Kriegsheim, Alexander; Humphries, Martin J; Norman, Jim C; Caswell, Patrick T

2013-09-16

Inhibition of αvβ3 or expression of mutant p53 promotes invasion into fibronectin (FN)-containing extracellular matrix (ECM) by enhancing Rab-coupling protein (RCP)-dependent recycling of α5β1 integrin. RCP and α5β1 cooperatively recruit receptor tyrosine kinases, including EGFR1, to regulate their trafficking and downstream signaling via protein kinase B (PKB)/Akt, which, in turn, promotes invasive migration. In this paper, we identify a novel PKB/Akt substrate, RacGAP1, which is phosphorylated as a consequence of RCP-dependent α5β1 trafficking. Phosphorylation of RacGAP1 promotes its recruitment to IQGAP1 at the tips of invasive pseudopods, and RacGAP1 then locally suppresses the activity of the cytoskeletal regulator Rac and promotes the activity of RhoA in this subcellular region. This Rac to RhoA switch promotes the extension of pseudopodial processes and invasive migration into FN-containing matrices, in a RhoA-dependent manner. Thus, the localized endocytic trafficking of α5β1 within the tips of invasive pseudopods elicits signals that promote the reorganization of the actin cytoskeleton, protrusion, and invasion into FN-rich ECM.

RCP-driven α5β1 recycling suppresses Rac and promotes RhoA activity via the RacGAP1–IQGAP1 complex

PubMed Central

Jacquemet, Guillaume; Green, David M.; Bridgewater, Rebecca E.; von Kriegsheim, Alexander; Humphries, Martin J.; Norman, Jim C.

2013-01-01

Inhibition of αvβ3 or expression of mutant p53 promotes invasion into fibronectin (FN)-containing extracellular matrix (ECM) by enhancing Rab-coupling protein (RCP)–dependent recycling of α5β1 integrin. RCP and α5β1 cooperatively recruit receptor tyrosine kinases, including EGFR1, to regulate their trafficking and downstream signaling via protein kinase B (PKB)/Akt, which, in turn, promotes invasive migration. In this paper, we identify a novel PKB/Akt substrate, RacGAP1, which is phosphorylated as a consequence of RCP-dependent α5β1 trafficking. Phosphorylation of RacGAP1 promotes its recruitment to IQGAP1 at the tips of invasive pseudopods, and RacGAP1 then locally suppresses the activity of the cytoskeletal regulator Rac and promotes the activity of RhoA in this subcellular region. This Rac to RhoA switch promotes the extension of pseudopodial processes and invasive migration into FN-containing matrices, in a RhoA-dependent manner. Thus, the localized endocytic trafficking of α5β1 within the tips of invasive pseudopods elicits signals that promote the reorganization of the actin cytoskeleton, protrusion, and invasion into FN-rich ECM. PMID:24019536

RhoGAP18B Isoforms Act on Distinct Rho-Family GTPases and Regulate Behavioral Responses to Alcohol via Cofilin

PubMed Central

Kalahasti, Geetha; Rodan, Aylin R.; Rothenfluh, Adrian

2015-01-01

Responses to the effects of ethanol are highly conserved across organisms, with reduced responses to the sedating effects of ethanol being predictive of increased risk for human alcohol dependence. Previously, we described that regulators of actin dynamics, such as the Rho-family GTPases Rac1, Rho1, and Cdc42, alter Drosophila’s sensitivity to ethanol-induced sedation. The GTPase activating protein RhoGAP18B also affects sensitivity to ethanol. To better understand how different RhoGAP18B isoforms affect ethanol sedation, we examined them for their effects on cell shape, GTP-loading of Rho-family GTPase, activation of the actin-severing cofilin, and actin filamentation. Our results suggest that the RhoGAP18B-PA isoform acts on Cdc42, while PC and PD act via Rac1 and Rho1 to activate cofilin. In vivo, a loss-of-function mutation in the cofilin-encoding gene twinstar leads to reduced ethanol-sensitivity and acts in concert with RhoGAP18B. Different RhoGAP18B isoforms, therefore, act on distinct subsets of Rho-family GTPases to modulate cofilin activity, actin dynamics, and ethanol-induced behaviors. PMID:26366560

Probing Conformational Changes of Human DNA Polymerase λ Using Mass Spectrometry-Based Protein Footprinting

PubMed Central

Fowler, Jason D.; Brown, Jessica A.; Kvaratskhelia, Mamuka; Suo, Zucai

2009-01-01

SUMMARY Crystallographic studies of the C-terminal, DNA polymerase β-like domain of human DNA polymerase lambda (fPolλ) suggested that the catalytic cycle might not involve a large protein domain rearrangement as observed with several replicative DNA polymerases and DNA polymerase β. To examine solution-phase protein conformation changes in fPolλ, which also contains a breast cancer susceptibility gene 1 C-terminal domain and a Proline-rich domain at its N-terminus, we used a mass spectrometry - based protein footprinting approach. In parallel experiments, surface accessibility maps for Arg residues were compared for the free fPolλ versus the binary complex of enzyme•gapped DNA and the ternary complex of enzyme•gapped DNA•dNTP. These experiments suggested that fPolλ does not undergo major conformational changes during the catalysis in the solution phase. Furthermore, the mass spectrometry-based protein footprinting experiments revealed that active site residue R386 was shielded from the surface only in the presence of both a gapped DNA substrate and an incoming nucleotide dNTP. Site-directed mutagenesis and pre-steady state kinetic studies confirmed the importance of R386 for the enzyme activity, and indicated the key role for its guanidino group in stabilizing the negative charges of an incoming nucleotide and the leaving pyrophosphate product. We suggest that such interactions could be shared by and important for catalytic functions of other DNA polymerases. PMID:19467241

Mechanisms of pertussis toxin-induced barrier dysfunction in bovine pulmonary artery endothelial cell monolayers.

PubMed

Patterson, C E; Stasek, J E; Schaphorst, K L; Davis, H W; Garcia, J G

1995-06-01

We have previously characterized several G proteins in endothelial cells (EC) as substrates for the ADP-ribosyltransferase activity of both pertussis (PT) and cholera toxin and described the modulation of key EC physiological responses, including gap formation and barrier function, by these toxins. In this study, we investigated the mechanisms involved in PT-mediated regulation of bovine pulmonary artery endothelial cells barrier function. PT caused a dose-dependent increase in albumin transfer, dependent upon action of the holotoxin, since neither the heat-inactivated PT, the isolated oligomer, nor the protomer induced EC permeability. PT-induced gap formation and barrier dysfunction were additive to either thrombin- or thrombin receptor-activating peptide-induced permeability, suggesting that thrombin and PT utilize distinct mechanisms. PT did not result in Ca2+ mobilization or alter either basal or thrombin-induced myosin light chain phosphorylation. However, PT stimulated protein kinase C (PKC) activation, and both PKC downregulation and PKC inhibition attenuated PT-induced permeability, indicating that PKC activity is involved in PT-induced barrier dysfunction. Like thrombin-induced permeability, the PT effect was blocked by prior increases in adenosine 3',5'-cyclic monophosphate. Thus PT-catalyzed ADP-ribosylation of a G protein (possibly other than Gi) may regulate cytoskeletal protein interactions, leading to EC barrier dysfunction.

An Arabidopsis Ran-binding protein, AtRanBP1c, is a co-activator of Ran GTPase-activating protein and requires the C-terminus for its cytoplasmic localization

NASA Technical Reports Server (NTRS)

Kim, Soo-Hwan; Roux, Stanley J.

2003-01-01

Ran-binding proteins (RanBPs) are a group of proteins that bind to Ran (Ras-related nuclear small GTP-binding protein), and thus either control the GTP/GDP-bound states of Ran or help couple the Ran GTPase cycle to a cellular process. AtRanBP1c is a Ran-binding protein from Arabidopsis thaliana (L.) Heynh. that was recently shown to be critically involved in the regulation of auxin-induced mitotic progression [S.-H. Kim et al. (2001) Plant Cell 13:2619-2630]. Here we report that AtRanBP1c inhibits the EDTA-induced release of GTP from Ran and serves as a co-activator of Ran-GTPase-activating protein (RanGAP) in vitro. Transient expression of AtRanBP1c fused to a beta-glucuronidase (GUS) reporter reveals that the protein localizes primarily to the cytosol. Neither the N- nor C-terminus of AtRanBP1c, which flank the Ran-binding domain (RanBD), is necessary for the binding of PsRan1-GTP to the protein, but both are needed for the cytosolic localization of GUS-fused AtRanBP1c. These findings, together with a previous report that AtRanBP1c is critically involved in root growth and development, imply that the promotion of GTP hydrolysis by the Ran/RanGAP/AtRanBP1c complex in the cytoplasm, and the resulting concentration gradient of Ran-GDP to Ran-GTP across the nuclear membrane could be important in the regulation of auxin-induced mitotic progression in root tips of A. thaliana.

Neuropathy-induced spinal GAP-43 expression is not a main player in the onset of mechanical pain hypersensitivity.

PubMed

Jaken, Robby J; van Gorp, Sebastiaan; Joosten, Elbert A; Losen, Mario; Martínez-Martínez, Pilar; De Baets, Marc; Marcus, Marco A; Deumens, Ronald

2011-12-01

Structural plasticity within the spinal nociceptive network may be fundamental to the chronic nature of neuropathic pain. In the present study, the spatiotemporal expression of growth-associated protein-43 (GAP-43), a protein which has been traditionally implicated in nerve fiber growth and sprouting, was investigated in relation to mechanical pain hypersensitivity. An L5 spinal nerve transection model was validated by the presence of mechanical pain hypersensitivity and an increase in the early neuronal activation marker cFos within the superficial spinal dorsal horn upon innocuous hindpaw stimulation. Spinal GAP-43 was found to be upregulated in the superficial L5 dorsal horn from 5 up to 10 days after injury. GAP-43 was co-localized with calcitonin-gene related peptide (CGRP), but not vesicular glutamate transporter-1 (VGLUT-1), IB4, or protein kinase-γ (PKC-γ), suggesting the regulation of GAP-43 in peptidergic nociceptive afferents. These GAP-43/CGRP fibers may be indicative of sprouting peptidergic fibers. Fiber sprouting largely depends on growth factors, which are typically associated with neuro-inflammatory processes. The putative role of neuropathy-induced GAP-43 expression in the development of mechanical pain hypersensitivity was investigated using the immune modulator propentofylline. Propentofylline treatment strongly attenuated the development of mechanical pain hypersensitivity and glial responses to nerve injury as measured by microglial and astroglial markers, but did not affect neuropathy-induced levels of spinal GAP-43 or GAP-43 regulation in CGRP fibers. We conclude that nerve injury induces structural plasticity in fibers expressing CGRP, which is regarded as a main player in central sensitization. Our data do not, however, support a major role of these structural changes in the onset of mechanical pain hypersensitivity.

Connexin 39.9 Protein Is Necessary for Coordinated Activation of Slow-twitch Muscle and Normal Behavior in Zebrafish*

PubMed Central

Hirata, Hiromi; Wen, Hua; Kawakami, Yu; Naganawa, Yuriko; Ogino, Kazutoyo; Yamada, Kenta; Saint-Amant, Louis; Low, Sean E.; Cui, Wilson W.; Zhou, Weibin; Sprague, Shawn M.; Asakawa, Kazuhide; Muto, Akira; Kawakami, Koichi; Kuwada, John Y.

2012-01-01

In many tissues and organs, connexin proteins assemble between neighboring cells to form gap junctions. These gap junctions facilitate direct intercellular communication between adjoining cells, allowing for the transmission of both chemical and electrical signals. In rodents, gap junctions are found in differentiating myoblasts and are important for myogenesis. Although gap junctions were once believed to be absent from differentiated skeletal muscle in mammals, recent studies in teleosts revealed that differentiated muscle does express connexins and is electrically coupled, at least at the larval stage. These findings raised questions regarding the functional significance of gap junctions in differentiated muscle. Our analysis of gap junctions in muscle began with the isolation of a zebrafish motor mutant that displayed weak coiling at day 1 of development, a behavior known to be driven by slow-twitch muscle (slow muscle). We identified a missense mutation in the gene encoding Connexin 39.9. In situ hybridization found connexin 39.9 to be expressed by slow muscle. Paired muscle recordings uncovered that wild-type slow muscles are electrically coupled, whereas mutant slow muscles are not. The further examination of cellular activity revealed aberrant, arrhythmic touch-evoked Ca2+ transients in mutant slow muscle and a reduction in the number of muscle fibers contracting in response to touch in mutants. These results indicate that Connexin 39.9 facilitates the spreading of neuronal inputs, which is irregular during motor development, beyond the muscle cells and that gap junctions play an essential role in the efficient recruitment of slow muscle fibers. PMID:22075003

Involvement of connexin 43 phosphorylation and gap junctional communication between smooth muscle cells in vasopressin-induced ROCK-dependent vasoconstriction after hemorrhagic shock.

PubMed

Yang, Guangming; Peng, Xiaoyong; Wu, Yue; Li, Tao; Liu, Liangming

2017-10-01

We examined the roles played by gap junctions (GJs) and the GJ channel protein connexin 43 (Cx43) in arginine vasopressin (AVP)-induced vasoconstriction after hemorrhagic shock and their relationship to Rho kinase (ROCK) and protein kinase C (PKC). The results showed that AVP induced an endothelium-independent contraction in rat superior mesenteric arteries (SMAs). Blocking the GJs significantly decreased the contractile response of SMAs and vascular smooth muscle cells (VSMCs) to AVP after shock and hypoxia. The selective Cx43-mimetic peptide inhibited the vascular contractile effect of AVP after shock and hypoxia. AVP restored hypoxia-induced decrease of Cx43 phosphorylation at Ser 262 and gap junctional communication in VSMCs. Activation of RhoA with U-46619 increased the contractile effect of AVP. This effect was antagonized by the ROCK inhibitor Y27632 and the Cx43-mimetic peptide. In contrast, neither an agonist nor an inhibitor of PKC had significant effects on AVP-induced contraction after hemorrhagic shock. In addition, silencing of Cx43 with siRNA blocked the AVP-induced increase of ROCK activity in hypoxic VSMCs. In conclusion, AVP-mediated vascular contractile effects are endothelium and myoendothelial gap junction independent. Gap junctions between VSMCs, gap junctional communication, and Cx43 phosphorylation at Ser 262 play important roles in the vascular effects of AVP. RhoA/ROCK, but not PKC, is involved in this process. Copyright © 2017 the American Physiological Society.

PKC-mediated HuD-GAP43 pathway activation in a mouse model of antiretroviral painful neuropathy.

PubMed

Sanna, M D; Quattrone, A; Ghelardini, C; Galeotti, N

2014-03-01

Patients treated with nucleoside reverse transcriptase inhibitors (NRTIs) develop painful neuropathies that lead to discontinuation of antiretroviral therapy thus limiting viral suppression strategies. The mechanisms by which NRTIs contribute to the development of neuropathy are not known. In order to elucidate the mechanisms underlying this drug-induced neuropathy, we have characterized cellular events in the central nervous system following antiretroviral treatment. Systemic administration of the antiretroviral agent, 2',3'-dideoxycytidine (ddC) considerably increased the expression and phosphorylation of protein kinase C (PKC) γ and ɛ, enzymes highly involved in pain processes, within periaqueductal grey matter (PAG), and, to a lesser extent, within thalamus and prefrontal cortex. These events appeared in coincidence with thermal and mechanical allodynia, but PKC blockade did not prevent the antiretroviral-induced pain hypersensitivity, ruling out a major involvement of PKC in the ddC-induced nociceptive behaviour. An increased expression of GAP43, a marker of neuroregeneration, and decreased levels of ATF3, a marker of neuroregeneration, were detected in all brain areas. ddC treatment also increased the expression of HuD, a RNA-binding protein target of PKC known to stabilize GAP43 mRNA. Pharmacological blockade of PKC prevented HuD and GAP43 overexpression. Silencing of both PKCγ and HuD reduced GAP43 levels in control mice and prevented the ddC-induced GAP43 enhanced expression. Present findings illustrate the presence of a supraspinal PKC-mediated HuD-GAP43 pathway activated by ddC. Based on our results, we speculate that antiretroviral drugs may recruit the HuD-GAP43 pathway, potentially contributing to a response to the antiretroviral neuronal toxicity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Platelet-derived growth factor receptor mediates activation of ras through different signaling pathways in different cell types.

PubMed Central

Satoh, T; Fantl, W J; Escobedo, J A; Williams, L T; Kaziro, Y

1993-01-01

A series of pieces of evidence have shown that Ras protein acts as a transducer of the platelet-derived growth factor (PDGF) receptor-mediated signaling pathway: (i) formation of Ras.GTP is detected immediately on PDGF stimulation, and (ii) a dominant inhibitory mutant Ras, as well as a neutralizing anti-Ras antibody, can interfere with PDGF-induced responses. On the other hand, several signal transducing molecules including phosphatidylinositol 3-kinase (PI3-K), GTPase-activating protein (GAP), and phospholipase C gamma (PLC gamma) bind directly to the PDGF receptor and become tyrosine phosphorylated. Recently, it was shown that specific phosphorylated tyrosines of the PDGF receptor are responsible for interaction between the receptor and each signaling molecule. However, the roles of these signaling molecules have not been elucidated, and it remains unclear which molecules are implicated in the Ras pathway. In this study, we measured Ras activation in cell lines expressing mutant PDGF receptors that are deficient in coupling with specific molecules. In fibroblast CHO cells, a mutant receptor (Y708F/Y719F [PI3-K-binding sites]) was unable to stimulate Ras, whereas another mutant (Y739F [the GAP-binding site]) could do so, suggesting an indispensable role of PI3-K or a protein that binds to the same sites as PI3-K for PDGF-stimulated Ras activation. By contrast, both of the above mutants were capable of stimulating Ras protein in a pro-B-cell line, BaF3. Furthermore, a mutant receptor (Y977F/Y989F [PLC gamma-binding sites]) could fully activate Ras, and the direct activation of protein kinase C and calcium mobilization had almost no effect on the GDP/GTP state of Ras in this cell line. These results suggest that, in the pro-B-cell transfectants, each of the above pathways (PI3-K, GAP, and PLC gamma) can be eliminated without a loss of Ras activation. It remains unclear whether another unknown essential pathway which regulates Ras protein exists within BaF3 cells. Therefore, it is likely that several different PDGF receptor-mediated signaling pathways function upstream of Ras, and the extent of the contribution of each pathway for the regulation of Ras may differ among different cell types. Images PMID:8388543

Cloning and expression of the Aspergillus oryzae glucan 1,3-beta-glucosidase A (exgA) in Pichia pastoris.

PubMed

Boonvitthya, Nassapat; Tanapong, Phatrapan; Kanngan, Patcharaporn; Burapatana, Vorakan; Chulalaksananukul, Warawut

2012-10-01

The glucan 1,3-beta-glucosidase A gene (exgA) from Aspergillus oryzae and fused to the Saccharomyces cerevisiae signal peptide (α-factor) was expressed under the control of either a constitutive (GAP) or an inducible (AOX1) promoter in Pichia pastoris. A 1.4-fold higher extracellular enzyme activity (2 U/ml) was obtained using the AOX1 inducible expression system than with the GAP constitutive promoter (1.4 U/ml). The purified recombinant ExgA enzyme, with a yield of 10 mg protein/l culture supernatant, was about 40 kDa by SDS-PAGE analysis with a specific activity of 289 U/mg protein. The enzyme was optimally active at 35 °C and pH 5.0 and displayed a K(M) and V(max) of 0.56 mM and 10,042 μmol/(min mg protein), respectively, with p-nitrophenyl-β-D-glucopyranoside as the substrate. Moreover, it was tolerant to glucose inhibition with a K(i) of 365 mM.

A Screen for Novel Phosphoinositide 3-kinase Effector Proteins*

PubMed Central

Dixon, Miles J.; Gray, Alexander; Boisvert, François-Michel; Agacan, Mark; Morrice, Nicholas A.; Gourlay, Robert; Leslie, Nicholas R.; Downes, C. Peter; Batty, Ian H.

2011-01-01

Class I phosphoinositide 3-kinases exert important cellular effects through their two primary lipid products, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). As few molecular targets for PtdIns(3,4)P2 have yet been identified, a screen for PI 3-kinase-responsive proteins that is selective for these is described. This features a tertiary approach incorporating a unique, primary recruitment of target proteins in intact cells to membranes selectively enriched in PtdIns(3,4)P2. A secondary purification of these proteins, optimized using tandem pleckstrin homology domain containing protein-1 (TAPP-1), an established PtdIns(3,4)P2 selective ligand, yields a fraction enriched in proteins of potentially similar lipid binding character that are identified by liquid chromatography-tandem MS. Thirdly, this approach is coupled to stable isotope labeling with amino acids in cell culture using differential isotope labeling of cells stimulated in the absence and presence of the PI 3-kinase inhibitor wortmannin. This provides a ratio-metric readout that distinguishes authentically responsive components from copurifying background proteins. Enriched fractions thus obtained from astrocytoma cells revealed a subset of proteins that exhibited ratios indicative of their initial, cellular responsiveness to PI 3-kinase activation. The inclusion among these of tandem pleckstrin homology domain containing protein-1, three isoforms of Akt, switch associated protein-70, early endosome antigen-1 and of additional proteins expressing recognized lipid binding domains demonstrates the utility of this strategy and lends credibility to the novel candidate proteins identified. The latter encompass a broad set of proteins that include the gene product of TBC1D2A, a putative Rab guanine nucleotide triphosphatase activating protein (GAP) and IQ motif containing GAP1, a potential tumor promoter. A sequence comparison of the former protein indicates the presence of a pleckstrin homology domain whose lipid binding character remains to be established. IQ motif containing GAP1 lacks known lipid interacting components and a preliminary analysis here indicates that this may exemplify a novel class of atypical phosphoinositide (aPI) binding domain. PMID:21263009

Mind the GAP: A Novel Tumor-Promoting Mechanism | Center for Cancer Research

Cancer.gov

RAS proteins, like light switches, toggle between an “on” conformation where they promote cell growth, survival, and/or the formation of blood vessels (known as angiogenesis) and an “off” conformation in which they are unable to stimulate their target effector proteins. Nearly one-third of human tumors express a mutated RAS gene, which encodes a protein locked permanently in the active state. Other tumors, including liver hepatocellular carcinomas (HCCs), display aberrant RAS pathway signaling but lack RAS gene mutations, suggesting alternative mechanisms for this excessive RAS activity.

Disruption of gap junctions attenuates aminoglycoside-elicited renal tubular cell injury.

PubMed

Yao, Jian; Huang, Tao; Fang, Xin; Chi, Yuan; Zhu, Ying; Wan, Yigang; Matsue, Hiroyuki; Kitamura, Masanori

2010-08-01

Gap junctions play important roles in the regulation of cell phenotype and in determining cell survival after various insults. Here, we investigated the role of gap junctions in aminoglycoside-induced injury to renal tubular cells. Two tubular epithelial cell lines NRK-E52 and LLC-PK1 were compared for gap junction protein expression and function by immunofluorescent staining, Western blot and dye transfer assay. Cell viability after exposure to aminoglycosides was evaluated by WST assay. Gap junctions were modulated by transfection of the gap junction protein, connexin 43 (Cx43), use of Cx43 siRNA and gap junction inhibitors. NRK-E52 cells expressed abundant Cx43 and were functionally coupled by gap junctional intercellular communication (GJIC). Exposure of NRK-E52 cells to aminoglycosides, G418 and hygromycin, increased Cx43 phosphorylation and GJIC. The aminoglycosides also decreased cell viability that was prevented by gap junction inhibitors and Cx43 siRNA. LLC-PK1 cells were gap junction-deficient and resistant to aminoglycoside-induced cytotoxicity. Over-expression of a wild-type Cx43 converted LLC-PK1 cells to a drug-sensitive phenotype. The gap junction inhibitor alpha-glycyrrhetinic acid (alpha-GA) activated Akt in NRK-E52 cells. Inhibition of the Akt pathway enhanced cell toxicity to G418 and abolished the protective effects of alpha-GA. In addition, gentamycin-elicited cytotoxicity in NRK-E52 cells was also significantly attenuated by alpha-GA. Gap junctions contributed to the cytotoxic effects of aminoglycosides. Modulation of gap junctions could be a promising approach for prevention and treatment of aminoglycoside-induced renal tubular cell injury.

Revisiting the Roco G-protein cycle.

PubMed

Terheyden, Susanne; Ho, Franz Y; Gilsbach, Bernd K; Wittinghofer, Alfred; Kortholt, Arjan

2015-01-01

Mutations in leucine-rich-repeat kinase 2 (LRRK2) are the most frequent cause of late-onset Parkinson's disease (PD). LRRK2 belongs to the Roco family of proteins which share a conserved Ras-like G-domain (Roc) and a C-terminal of Roc (COR) domain tandem. The nucleotide state of small G-proteins is strictly controlled by guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). Because of contradictory structural and biochemical data, the regulatory mechanism of the LRRK2 Roc G-domain and the RocCOR tandem is still under debate. In the present study, we solved the first nucleotide-bound Roc structure and used LRRK2 and bacterial Roco proteins to characterize the RocCOR function in more detail. Nucleotide binding induces a drastic structural change in the Roc/COR domain interface, a region strongly implicated in patients with an LRRK2 mutation. Our data confirm previous assumptions that the C-terminal subdomain of COR functions as a dimerization device. We show that the dimer formation is independent of nucleotide. The affinity for GDP/GTP is in the micromolar range, the result of which is high dissociation rates in the s-1 range. Thus Roco proteins are unlikely to need GEFs to achieve activation. Monomeric LRRK2 and Roco G-domains have a similar low GTPase activity to small G-proteins. We show that GTPase activity in bacterial Roco is stimulated by the nucleotide-dependent dimerization of the G-domain within the complex. We thus propose that the Roco proteins do not require GAPs to stimulate GTP hydrolysis but stimulate each other by one monomer completing the catalytic machinery of the other.

Review article: mitogen-activated protein kinases in chronic intestinal inflammation - targeting ancient pathways to treat modern diseases.

PubMed

Waetzig, G H; Schreiber, S

2003-07-01

Conventional treatment of chronic inflammatory disorders, including inflammatory bowel diseases, employs broad-range anti-inflammatory drugs. In order to reduce the side-effects and increase the efficacy of treatment, several strategies have been developed in the last decade to interfere with intercellular and intracellular inflammatory signalling processes. The highly conserved mitogen-activated protein kinase pathways regulate most cellular processes, particularly defence mechanisms such as stress reactions and inflammation. In this review, we provide an overview of the current knowledge of the specificity and interconnection of mitogen-activated protein kinase pathways, their functions in the gut immune system and published and ongoing studies on the role of mitogen-activated protein kinases in inflammatory bowel disease. The development of mitogen-activated protein kinase inhibitors and their use for the therapy of inflammatory disorders is a paradigm of the successful bridging of the gap between basic research and clinical practice.

The TAT-RasGAP317-326 anti-cancer peptide can kill in a caspase-, apoptosis-, and necroptosis-independent manner

PubMed Central

Puyal, Julien; Margue, Christiane; Michel, Sébastien; Kreis, Stephanie; Kulms, Dagmar; Barras, David; Nahimana, Aimable; Widmann, Christian

2016-01-01

Tumor cell resistance to apoptosis, which is triggered by many anti-tumor therapies, remains a major clinical problem. Therefore, development of more efficient therapies is a priority to improve cancer prognosis. We have previously shown that a cell-permeable peptide derived from the p120 Ras GTPase-activating protein (RasGAP), called TAT-RasGAP317-326, bears anti-malignant activities in vitro and in vivo, such as inhibition of metastatic progression and tumor cell sensitization to cell death induced by various anti-cancer treatments. Recently, we discovered that this RasGAP-derived peptide possesses the ability to directly kill some cancer cells. TAT-RasGAP317-326 can cause cell death in a manner that can be either partially caspase-dependent or fully caspase-independent. Indeed, TAT-RasGAP317-326-induced toxicity was not or only partially prevented when apoptosis was inhibited. Moreover, blocking other forms of cell death, such as necroptosis, parthanatos, pyroptosis and autophagy did not hamper the killing activity of the peptide. The death induced by TAT-RasGAP317-326 can therefore proceed independently from these modes of death. Our finding has potentially interesting clinical relevance because activation of a death pathway that is distinct from apoptosis and necroptosis in tumor cells could lead to the generation of anti-cancer drugs that target pathways not yet considered for cancer treatment. PMID:27602963

HIV-tat alters Connexin43 expression and trafficking in human astrocytes: role in NeuroAIDS.

PubMed

Berman, Joan W; Carvallo, Loreto; Buckner, Clarisa M; Luers, Aimée; Prevedel, Lisa; Bennett, Michael V; Eugenin, Eliseo A

2016-03-02

HIV-associated neurocognitive disorders (HAND) are a major complication in at least half of the infected population despite effective antiretroviral treatment and immune reconstitution. HIV-associated CNS damage is not correlated with active viral replication but instead is associated with mechanisms that regulate inflammation and neuronal compromise. Our data indicate that one of these mechanisms is mediated by gap junction channels and/or hemichannels. Normally, gap junction channels shutdown under inflammatory conditions, including viral diseases. However, HIV infection upregulates Connexin43 (Cx43) expression and maintains gap junctional communication by unknown mechanism(s). Human primary astrocytes were exposed to several HIV proteins as well as to HIV, and expression and function of Connexin43- and Connexin30-containing channels were determined by western blot, immunofluorescence, microinjection of a fluorescent tracer and chromatin immunoprecipitation (ChIP). Here, we demonstrate that HIV infection increases Cx43 expression in vivo. HIV-tat, the transactivator of the virus, and no other HIV proteins tested, increases Cx43 expression and maintains functional gap junctional communication in human astrocytes. Cx43 upregulation is mediated by binding of the HIV-tat protein to the Cx43 promoter, but not to the Cx30 promoter, resulting in increased Cx43 messenger RNA (mRNA) and protein as well as gap junctional communication. We propose that HIV-tat contributes to the spread of intracellular toxic signals generated in a few HIV-infected cells into surrounding uninfected cells by upregulating gap junctional communication. In the current antiretroviral era, where HIV replication is often completely suppressed, viral factors such as HIV-tat are still produced and released from infected cells. Thus, blocking the effects of HIV-tat could result in new strategies to reduce the damaging consequences of HIV infection of the CNS.

Dissection of neuronal gap junction circuits that regulate social behavior in Caenorhabditis elegans.

PubMed

Jang, Heeun; Levy, Sagi; Flavell, Steven W; Mende, Fanny; Latham, Richard; Zimmer, Manuel; Bargmann, Cornelia I

2017-02-14

A hub-and-spoke circuit of neurons connected by gap junctions controls aggregation behavior and related behavioral responses to oxygen, pheromones, and food in Caenorhabditis elegans The molecular composition of the gap junctions connecting RMG hub neurons with sensory spoke neurons is unknown. We show here that the innexin gene unc-9 is required in RMG hub neurons to drive aggregation and related behaviors, indicating that UNC-9-containing gap junctions mediate RMG signaling. To dissect the circuit in detail, we developed methods to inhibit unc-9 -based gap junctions with dominant-negative unc-1 transgenes. unc-1(dn) alters a stomatin-like protein that regulates unc-9 electrical signaling; its disruptive effects can be rescued by a constitutively active UNC-9::GFP protein, demonstrating specificity. Expression of unc-1(dn) in RMG hub neurons, ADL or ASK pheromone-sensing neurons, or URX oxygen-sensing neurons disrupts specific elements of aggregation-related behaviors. In ADL, unc-1(dn) has effects opposite to those of tetanus toxin light chain, separating the roles of ADL electrical and chemical synapses. These results reveal roles of gap junctions in a complex behavior at cellular resolution and provide a tool for similar exploration of other gap junction circuits.

Dissection of neuronal gap junction circuits that regulate social behavior in Caenorhabditis elegans

PubMed Central

Jang, Heeun; Levy, Sagi; Flavell, Steven W.; Mende, Fanny; Latham, Richard; Zimmer, Manuel; Bargmann, Cornelia I.

2017-01-01

A hub-and-spoke circuit of neurons connected by gap junctions controls aggregation behavior and related behavioral responses to oxygen, pheromones, and food in Caenorhabditis elegans. The molecular composition of the gap junctions connecting RMG hub neurons with sensory spoke neurons is unknown. We show here that the innexin gene unc-9 is required in RMG hub neurons to drive aggregation and related behaviors, indicating that UNC-9–containing gap junctions mediate RMG signaling. To dissect the circuit in detail, we developed methods to inhibit unc-9–based gap junctions with dominant-negative unc-1 transgenes. unc-1(dn) alters a stomatin-like protein that regulates unc-9 electrical signaling; its disruptive effects can be rescued by a constitutively active UNC-9::GFP protein, demonstrating specificity. Expression of unc-1(dn) in RMG hub neurons, ADL or ASK pheromone-sensing neurons, or URX oxygen-sensing neurons disrupts specific elements of aggregation-related behaviors. In ADL, unc-1(dn) has effects opposite to those of tetanus toxin light chain, separating the roles of ADL electrical and chemical synapses. These results reveal roles of gap junctions in a complex behavior at cellular resolution and provide a tool for similar exploration of other gap junction circuits. PMID:28143932

Identification of the neurofibromatosis type 1 gene product

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gutmann, D.H.; Wood, D.L.; Collins, F.S.

The gene for neurofibromatosis type 1 (NF1) was recently identified by positional cloning. The complete cDNA encodes a polypeptide of 2818 amino acids. To study the NF1 gene product, antibodies were raised against both fusion proteins and synthetic peptides. Initial characterization of two anti-peptide antibodies and one fusion-protein antibody demonstrated a specific protein of {approx}250 kDa by both immunoprecipitation and immunoblotting. This protein was found in all tissues and cell lines examined and is detected in human, rat, and mouse tissues. To demonstrate that these antibodies specifically recognize the NF1 protein, additional fusion proteins containing the sequence specific to themore » synthetic peptide were generated. Both peptide antisera recognize the proper specific fusion proteins so generated. Immunoprecipitates using the peptide antisera were shown to recognize the same protein detected by immunoblotting with either the other peptide antiserum or the fusion-protein antiserum. Immunoblotting using antiserum specific to spatially distinct epitopes conducted on tissue homogenates demonstrated the NF1 protein in all adult tissues. Based on the homology between the NF1 gene product and members of the GTPase-activating protein (GAP) superfamily, the name NF1-GAP-related protein (NF1GRP) is suggested.« less

Klebsiella aerogenes UreF: Identification of the UreG Binding Site and Role in Enhancing the Fidelity of Urease Activation†

PubMed Central

Boer, Jodi L.; Hausinger, Robert P.

2012-01-01

The Ni-containing active site of Klebsiella aerogenes urease is assembled through the concerted action of the UreD, UreE, UreF, and UreG accessory proteins. UreE functions as a metallochaperone that delivers Ni to a complex of UreD—UreF—UreG bound to urease apoprotein, with UreG serving as a GTPase during enzyme activation. The present study focuses on the role of UreF, previously proposed to act as a GTPase activating protein (GAP) of UreG. Sixteen conserved UreF surface residues that may play roles in protein:protein interactions were independently changed to Ala. When produced in the context of the entire urease gene cluster, cell-free extracts of nine site-directed mutants had less than 10% of the wild-type urease activity. Enrichment of the variant forms of UreF, as the UreE-F fusion proteins, uniformly resulted in co-purification of UreD and urease apoprotein; whereas UreG bound to only a subset of the species. Notably, reduced interaction with UreG correlated with the low activity mutants. The affected residues in UreF map to a distinct surface on the crystal structure, defining the UreG binding site. In contrast to the hypothesis that UreF is a GAP, the UreD—UreF—UreG—urease apoprotein complex containing K165A UreF exhibited significantly greater levels of GTPase activity than that containing the wild-type protein. Additional studies demonstrated the UreG GTPase activity was largely uncoupled from urease activation for the complex containing this UreF variant. Further experiments with these complexes provided evidence that UreF gates the GTPase activity of UreG to enhance the fidelity of urease metallocenter assembly, especially in the presence of the non-cognate metal Zn. PMID:22369361

Boldine Prevents Renal Alterations in Diabetic Rats

PubMed Central

Hernández-Salinas, Romina; Vielma, Alejandra Z.; Arismendi, Marlene N.; Boric, Mauricio P.; Sáez, Juan C.; Velarde, Victoria

2013-01-01

Diabetic nephropathy alters both structure and function of the kidney. These alterations are associated with increased levels of reactive oxygen species, matrix proteins, and proinflammatory molecules. Inflammation decreases gap junctional communication and increases hemichannel activity leading to increased membrane permeability and altering tissue homeostasis. Since current treatments for diabetic nephropathy do not prevent renal damage, we postulated an alternative treatment with boldine, an alkaloid obtained from boldo with antioxidant, anti-inflammatory, and hypoglycemic effects. Streptozotocin-induced diabetic and control rats were treated or not treated with boldine (50 mg/Kg/day) for ten weeks. In addition, mesangial cells were cultured under control conditions or in high glucose concentration plus proinflammatory cytokines, with or without boldine (100 µmol/L). Boldine treatment in diabetic animals prevented the increase in glycemia, blood pressure, renal thiobarbituric acid reactive substances and the urinary protein/creatinine ratio. Boldine also reduced alterations in matrix proteins and markers of renal damage. In mesangial cells, boldine prevented the increase in oxidative stress, the decrease in gap junctional communication, and the increase in cell permeability due to connexin hemichannel activity induced by high glucose and proinflammatory cytokines but did not block gap junction channels. Thus boldine prevented both renal and cellular alterations and could be useful for preventing tissue damage in diabetic subjects. PMID:24416726

Boldine prevents renal alterations in diabetic rats.

PubMed

Hernández-Salinas, Romina; Vielma, Alejandra Z; Arismendi, Marlene N; Boric, Mauricio P; Sáez, Juan C; Velarde, Victoria

2013-01-01

Diabetic nephropathy alters both structure and function of the kidney. These alterations are associated with increased levels of reactive oxygen species, matrix proteins, and proinflammatory molecules. Inflammation decreases gap junctional communication and increases hemichannel activity leading to increased membrane permeability and altering tissue homeostasis. Since current treatments for diabetic nephropathy do not prevent renal damage, we postulated an alternative treatment with boldine, an alkaloid obtained from boldo with antioxidant, anti-inflammatory, and hypoglycemic effects. Streptozotocin-induced diabetic and control rats were treated or not treated with boldine (50 mg/Kg/day) for ten weeks. In addition, mesangial cells were cultured under control conditions or in high glucose concentration plus proinflammatory cytokines, with or without boldine (100 µmol/L). Boldine treatment in diabetic animals prevented the increase in glycemia, blood pressure, renal thiobarbituric acid reactive substances and the urinary protein/creatinine ratio. Boldine also reduced alterations in matrix proteins and markers of renal damage. In mesangial cells, boldine prevented the increase in oxidative stress, the decrease in gap junctional communication, and the increase in cell permeability due to connexin hemichannel activity induced by high glucose and proinflammatory cytokines but did not block gap junction channels. Thus boldine prevented both renal and cellular alterations and could be useful for preventing tissue damage in diabetic subjects.

Preparation of Gap Junctions in Membrane Microdomains for Immunoprecipitation and Mass Spectrometry Interactome Analysis.

PubMed

Fowler, Stephanie; Akins, Mark; Bennett, Steffany A L

2016-01-01

Protein interaction networks at gap junction plaques are increasingly implicated in a variety of intracellular signaling cascades. Identifying protein interactions of integral membrane proteins is a valuable tool for determining channel function. However, several technical challenges exist. Subcellular fractionation of the bait protein matrix is usually required to identify less abundant proteins in complex homogenates. Sufficient solvation of the lipid environment without perturbation of the protein interactome must also be achieved. The present chapter describes the flotation of light and heavy liver tissue membrane microdomains to facilitate the identification and analysis of endogenous gap junction proteins and includes technical notes for translation to other integral membrane proteins, tissues, or cell culture models. These procedures are valuable tools for the enrichment of gap junction membrane compartments and for the identification of gap junction signaling interactomes.

A gene delivery system containing nuclear localization signal: Increased nucleus import and transfection efficiency with the assistance of RanGAP1.

PubMed

Chen, Kang; Guo, Lingling; Zhang, Jiulong; Chen, Qing; Wang, Kuanglei; Li, Chenxi; Li, Weinan; Qiao, Mingxi; Zhao, Xiuli; Hu, Haiyang; Chen, Dawei

2017-01-15

In the present report, a degradable gene delivery system (PAMS/DNA/10NLS) containing nucleus location signal peptide (NLS) was prepared. The agarose gel electrophoresis, particle size and zeta potential of PAMS/DNA/10NLS were similar to those of PAMS/DNA, which proved that NLS did not affect the interaction between PAMS and DNA. PAMS/DNA/10NLS exhibited marked extracellular and intracellular degradation under acidic conditions. The degradation was believed to allow NLS to come into contact with importins easily, which was able to mediate the nucleus import. With the help of NLS, PAMS/DNA/10NLS exhibited a higher transfection capability than PAMS/DNA. Moreover, the transfection of PAMS/DNA/10NLS was less dependent on the breakdown of the nucleus envelope than PAMS/DNA. Considering that GTPase-activating protein 1 (RanGAP1) was able to activate the endogenous GTPase, which was necessary for NLS-mediated nucleus import, RanGAP1 overexpressed cells (RanGAP1 cells) were produced. This result showed that RanGAP1 cells had higher GTPase activities than normal cells. Both the nucleus import and transfection efficiency of PAMS/DNA/10NLS were markedly higher in RanGAP1 cells than that in normal cells. The in vivo transfection results also showed that the transfection efficiency of PAMS/DNA/10NLS was higher in RanGAP1 pre-treated mice than that in normal mice. These findings showed that PAMS/DNA/10NLS is a promising gene delivery system with the assistance of RanGAP1. The present report describes the increased transfection efficiency of a degradable gene delivery system (PAMS/DNA/10NLS) containing nuclear location signal (NLS) with the assistance of GTPase-activating protein 1 (RanGAP1). The physicochemical properties of PAMS/DNA/10NLS were similar to those of PAMS/DNA. PAMS/DNA/10NLS exhibited great extracellular and intracellular degradations, which might allow NLS to contact with importins easily. With the help of NLS, PAMS/DNA/10NLS exhibited a higher transfection capability than PAMS/DNA. The transfection of PAMS/DNA/10NLS had less dependence on the breakdown of nuclear envelope. Both the nuclear import and transfection efficiency of PAMS/DNA/10NLS were higher in RanGAP1 overexpressed cells than that in normal cells. Moreover, the transfection efficiency of PAMS/DNA/10NLS was higher in RanGAP1 pre-treated mice than that in normal mice. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A single splice site mutation in human-specific ARHGAP11B causes basal progenitor amplification

PubMed Central

Florio, Marta; Namba, Takashi; Pääbo, Svante; Hiller, Michael; Huttner, Wieland B.

2016-01-01

The gene ARHGAP11B promotes basal progenitor amplification and is implicated in neocortex expansion. It arose on the human evolutionary lineage by partial duplication of ARHGAP11A, which encodes a Rho guanosine triphosphatase–activating protein (RhoGAP). However, a lack of 55 nucleotides in ARHGAP11B mRNA leads to loss of RhoGAP activity by GAP domain truncation and addition of a human-specific carboxy-terminal amino acid sequence. We show that these 55 nucleotides are deleted by mRNA splicing due to a single C→G substitution that creates a novel splice donor site. We reconstructed an ancestral ARHGAP11B complementary DNA without this substitution. Ancestral ARHGAP11B exhibits RhoGAP activity but has no ability to increase basal progenitors during neocortex development. Hence, a single nucleotide substitution underlies the specific properties of ARHGAP11B that likely contributed to the evolutionary expansion of the human neocortex. PMID:27957544

Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP.

PubMed

Yu, Qin; Hu, Liyan; Yao, Qing; Zhu, Yongqun; Dong, Na; Wang, Da-Cheng; Shao, Feng

2013-06-01

Rab GTPases are emerging targets of diverse bacterial pathogens. Here, we perform biochemical and structural analyses of LepB, a Rab GTPase-activating protein (GAP) effector from Legionella pneumophila. We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors. Exhaustive mutation analyses identify Arg444 as the arginine finger, but no catalytically essential glutamine residues. Crystal structures of LepB313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AlF3 support the catalytic role of Arg444, and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs. Glu449, structurally equivalent to TBC RabGAP glutamine finger in apo-LepB, undergoes a drastic movement upon Rab1 binding, which induces Rab1 Gln70 side-chain flipping towards GDP-AlF3 through a strong ionic interaction. This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB. Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.

Expression of endoglucanases in Pichia pastoris under control of the GAP promoter

PubMed Central

2014-01-01

Background Plant-derived biomass is a potential alternative to fossil feedstocks for a greener economy. Enzymatic saccharification of biomass has been studied extensively and endoglucanases have been found to be a prerequisite for quick initial liquefaction of biomass under industrial conditions. Pichia pastoris, widely used for heterologous protein expression, can be utilized for fungal endoglucanase production. The recently marketed PichiaPink™ expression system allows for rapid clone selection, and employs the methanol inducible AOX1 promoter to ensure high protein expression levels. However, methanol is toxic and poses a fire hazard, issues which become more significant at an industrial scale. It is possible to eliminate these risks and still maintain high productivity by switching to the constitutive GAP promoter. Results In the present study, a plasmid carrying the constitutive GAP promoter was created for PichiaPink™. We then studied expression of two endoglucanases, AfCel12A from Aspergillus fumigatus and TaCel5A from Thermoascus aurantiacus, regulated by either the AOX1 promoter or the GAP promoter. Initial experiments in tubes and small bioreactors showed that the levels of AfCel12A obtained with the constitutive promoter were similar or higher, compared to the AOX1 promoter, whereas the levels of TaCel5A were somewhat lower. After optimization of cultivation conditions using a 15-l bioreactor, the recombinant P. pastoris strains utilizing the GAP promoter produced ca. 3–5 g/l of total secreted protein, with CMCase activity equivalent to 1200 nkat/ml AfCel12A and 170 nkat/ml TaCel5A. Conclusions We present a strategy for constitutive recombinant protein expression in the novel PichiaPink™ system. Both AfCel12A and TaCel5A were successfully expressed constitutively in P. pastoris under the GAP promoter. Reasonable protein levels were reached after optimizing cultivation conditions. PMID:24742273

Expression of endoglucanases in Pichia pastoris under control of the GAP promoter.

PubMed

Várnai, Anikó; Tang, Campbell; Bengtsson, Oskar; Atterton, Andrew; Mathiesen, Geir; Eijsink, Vincent G H

2014-04-18

Plant-derived biomass is a potential alternative to fossil feedstocks for a greener economy. Enzymatic saccharification of biomass has been studied extensively and endoglucanases have been found to be a prerequisite for quick initial liquefaction of biomass under industrial conditions. Pichia pastoris, widely used for heterologous protein expression, can be utilized for fungal endoglucanase production. The recently marketed PichiaPink™ expression system allows for rapid clone selection, and employs the methanol inducible AOX1 promoter to ensure high protein expression levels. However, methanol is toxic and poses a fire hazard, issues which become more significant at an industrial scale. It is possible to eliminate these risks and still maintain high productivity by switching to the constitutive GAP promoter. In the present study, a plasmid carrying the constitutive GAP promoter was created for PichiaPink™. We then studied expression of two endoglucanases, AfCel12A from Aspergillus fumigatus and TaCel5A from Thermoascus aurantiacus, regulated by either the AOX1 promoter or the GAP promoter. Initial experiments in tubes and small bioreactors showed that the levels of AfCel12A obtained with the constitutive promoter were similar or higher, compared to the AOX1 promoter, whereas the levels of TaCel5A were somewhat lower. After optimization of cultivation conditions using a 15-l bioreactor, the recombinant P. pastoris strains utilizing the GAP promoter produced ca. 3-5 g/l of total secreted protein, with CMCase activity equivalent to 1200 nkat/ml AfCel12A and 170 nkat/ml TaCel5A. We present a strategy for constitutive recombinant protein expression in the novel PichiaPink™ system. Both AfCel12A and TaCel5A were successfully expressed constitutively in P. pastoris under the GAP promoter. Reasonable protein levels were reached after optimizing cultivation conditions.

An ADP-Ribosylation Factor GTPase-activating Protein Git2-short/KIAA0148 Is Involved in Subcellular Localization of Paxillin and Actin Cytoskeletal Organization

PubMed Central

Mazaki, Yuichi; Hashimoto, Shigeru; Okawa, Katsuya; Tsubouchi, Asako; Nakamura, Kuniaki; Yagi, Ryohei; Yano, Hajime; Kondo, Akiko; Iwamatsu, Akihiro; Mizoguchi, Akira; Sabe, Hisataka

2001-01-01

Paxillin acts as an adaptor protein in integrin signaling. We have shown that paxillin exists in a relatively large cytoplasmic pool, including perinuclear areas, in addition to focal complexes formed at the cell periphery and focal adhesions formed underneath the cell. Several ADP-ribosylation factor (ARF) GTPase-activating proteins (GAPs; ARFGAPs) have been shown to associate with paxillin. We report here that Git2-short/KIAA0148 exhibits properties of a paxillin-associated ARFGAP and appears to be colocalized with paxillin, primarily at perinuclear areas. A fraction of Git2-short was also localized to actin-rich structures at the cell periphery. Unlike paxillin, however, Git2-short did not accumulate at focal adhesions underneath the cell. Git2-short is a short isoform of Git2, which is highly homologous to p95PKL, another paxillin-binding protein, and showed a weaker binding affinity toward paxillin than that of Git2. The ARFGAP activities of Git2 and Git2-short have been previously demonstrated in vitro, and we provided evidence that at least one ARF isoform, ARF1, is an intracellular substrate for the GAP activity of Git2-short. We also showed that Git2-short could antagonize several known ARF1-mediated phenotypes: overexpression of Git2-short, but not its GAP-inactive mutant, caused the redistribution of Golgi protein β-COP and reduced the amounts of paxillin-containing focal adhesions and actin stress fibers. Perinuclear localization of paxillin, which was sensitive to ARF inactivation, was also affected by Git2-short overexpression. On the other hand, paxillin localization to focal complexes at the cell periphery was unaffected or even augmented by Git2-short overexpression. Therefore, an ARFGAP protein weakly interacting with paxillin, Git2-short, exhibits pleiotropic functions involving the regulation of Golgi organization, actin cytoskeletal organization, and subcellular localization of paxillin, all of which need to be coordinately regulated during integrin-mediated cell adhesion and intracellular signaling. PMID:11251077

Genetic Screening in C. Elegans Identifies Rho-GTPAse Activating Protein 6 as Novel HERG Regulator

PubMed Central

Potet, Franck; Petersen, Christina I.; Boutaud, Olivier; Shuai, Wen; Stepanovic, Svetlana Z.; Balser, Jeffrey R.; Kupershmidt, Sabina

2009-01-01

The human ether-a-go-go related gene (HERG) constitutes the pore forming subunit of IKr, a K+ current involved in repolarization of the cardiac action potential. While mutations in HERG predispose patients to cardiac arrhythmias (Long QT syndrome; LQTS), altered function of HERG regulators are undoubtedly LQTS risk factors. We have combined RNA interference with behavioral screening in Caenorhabditis elegans to detect genes that influence function of the HERG homolog, UNC-103. One such gene encodes the worm ortholog of the rho-GTPase activating protein 6 (ARHGAP6). In addition to its GAP function, ARHGAP6 induces cytoskeletal rearrangements and activates phospholipase C (PLC). Here we show that IKr recorded in cells co-expressing HERG and ARHGAP6 was decreased by 43% compared to HERG alone. Biochemical measurements of cell-surface associated HERG revealed that ARHGAP6 reduced membrane expression of HERG by 35%, which correlates well with the reduction in current. In an atrial myocyte cell line, suppression of endogenous ARHGAP6 by virally transduced shRNA led to a 53 % enhancement of IKr. ARHGAP6 effects were maintained when we introduced a dominant negative rho-GTPase, or ARHGAP6 devoid of rhoGAP function, indicating ARHGAP6 regulation of HERG is independent of rho activation. However, ARHGAP6 lost effectiveness when PLC was inhibited. We further determined that ARHGAP6 effects are mediated by a consensus SH3 binding domain within the C-terminus of HERG, although stable ARHGAP6-HERG complexes were not observed. These data link a rhoGAP-activated PLC pathway to HERG membrane expression and implicate this family of proteins as candidate genes in disorders involving HERG. PMID:19038263

Activation of protein kinase C and disruption of endothelial monolayer integrity by sodium arsenite-Potential mechanism in the development of atherosclerosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pereira, Flavia E.; Coffin, J. Douglas; Beall, Howard D.

2007-04-15

Arsenic exposure has been shown to exacerbate atherosclerosis, beginning with activation of the endothelium that lines the vessel wall. Endothelial barrier integrity is maintained by proteins of the adherens junction (AJ) such as vascular endothelial cadherin (VE-cadherin) and {beta}-catenin and their association with the actin cytoskeleton. In the present study, human aortic endothelial cells (HAECs) were exposed to 1, 5 and 10 {mu}M sodium arsenite [As(III)] for 1, 6, 12 and 24 h, and the effects on endothelial barrier integrity were determined. Immunofluorescence studies revealed formation of actin stress fibers and non-uniform VE-cadherin and {beta}-catenin staining at cell-cell junctions thatmore » were concentration- and time-dependent. Intercellular gaps were observed with a measured increase in endothelial permeability. In addition, concentration-dependent increases in tyrosine phosphorylation (PY) of {beta}-catenin and activation of protein kinase C{alpha} (PKC{alpha}) were observed. Inhibition of PKC{alpha} restored VE-cadherin and {beta}-catenin staining at cell-cell junctions and abolished the As(III)-induced formation of actin stress fibers and intercellular gaps. Endothelial permeability and PY of {beta}-catenin were also reduced to basal levels. These results demonstrate that As(III) induces activation of PKC{alpha}, which leads to increased PY of {beta}-catenin downstream of PKC{alpha} activation. Phosphorylation of {beta}-catenin plausibly severs the association of VE-cadherin and {beta}-catenin, which along with formation of actin stress fibers, results in intercellular gap formation and increased endothelial permeability. To the best of our knowledge, this is the first report demonstrating that As(III) causes a loss of endothelial monolayer integrity, which potentially could contribute to the development of atherosclerosis.« less

Quantitative in vivo Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Toxoplasma Invasion Motor Complex

PubMed Central

Nebl, Thomas; Prieto, Judith Helena; Kapp, Eugene; Smith, Brian J.; Williams, Melanie J.; Yates, John R.; Cowman, Alan F.; Tonkin, Christopher J.

2011-01-01

Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca2+-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of 32[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca2+-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component. PMID:21980283

The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Kuo; Williams, C. David; McGill, Mitchell R.

2013-12-15

Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented whenmore » animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5 h after APAP. However, the protection was completely lost when 2-APB was given 4–6 h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions. - Highlights: • 2-APB protected against APAP-induced liver injury in mice in vivo and in vitro • 2-APB protected by inhibiting APAP metabolic activation and JNK signaling pathway • DMSO inhibited APAP metabolic activation as the solvent of 2-APB • Off-target effects of connexin32 gene knock-out mice need to be considered.« less

Disruption of gap junctions attenuates aminoglycoside-elicited renal tubular cell injury

PubMed Central

Yao, Jian; Huang, Tao; Fang, Xin; Chi, Yuan; Zhu, Ying; Wan, Yigang; Matsue, Hiroyuki; Kitamura, Masanori

2010-01-01

BACKGROUND AND PURPOSE Gap junctions play important roles in the regulation of cell phenotype and in determining cell survival after various insults. Here, we investigated the role of gap junctions in aminoglycoside-induced injury to renal tubular cells. EXPERIMENTAL APPROACH Two tubular epithelial cell lines NRK-E52 and LLC-PK1 were compared for gap junction protein expression and function by immunofluorescent staining, Western blot and dye transfer assay. Cell viability after exposure to aminoglycosides was evaluated by WST assay. Gap junctions were modulated by transfection of the gap junction protein, connexin 43 (Cx43), use of Cx43 siRNA and gap junction inhibitors. KEY RESULTS NRK-E52 cells expressed abundant Cx43 and were functionally coupled by gap junctional intercellular communication (GJIC). Exposure of NRK-E52 cells to aminoglycosides, G418 and hygromycin, increased Cx43 phosphorylation and GJIC. The aminoglycosides also decreased cell viability that was prevented by gap junction inhibitors and Cx43 siRNA. LLC-PK1 cells were gap junction-deficient and resistant to aminoglycoside-induced cytotoxicity. Over-expression of a wild-type Cx43 converted LLC-PK1 cells to a drug-sensitive phenotype. The gap junction inhibitor α-glycyrrhetinic acid (α-GA) activated Akt in NRK-E52 cells. Inhibition of the Akt pathway enhanced cell toxicity to G418 and abolished the protective effects of α-GA. In addition, gentamycin-elicited cytotoxicity in NRK-E52 cells was also significantly attenuated by α-GA. CONCLUSION AND IMPLICATIONS Gap junctions contributed to the cytotoxic effects of aminoglycosides. Modulation of gap junctions could be a promising approach for prevention and treatment of aminoglycoside-induced renal tubular cell injury. PMID:20649601

Control of neuronal morphology and connectivity: emerging developmental roles for gap junctional proteins.

PubMed

Baker, Michael W; Macagno, Eduardo R

2014-04-17

Recent evidence indicates that gap junction (GJ) proteins can play a critical role in controlling neuronal connectivity as well as cell morphology in the developing nervous system. GJ proteins may function analogously to cell adhesion molecules, mediating cellular recognition and selective neurite adhesion. Moreover, during synaptogenesis electrical synapses often herald the later establishment of chemical synapses, and thus may help facilitate activity-dependent sculpting of synaptic terminals. Recent findings suggest that the morphology and connectivity of embryonic leech neurons are fundamentally organized by the type and perhaps location of the GJ proteins they express. For example, ectopic expression in embryonic leech neurons of certain innexins that define small GJ-linked networks of cells leads to the novel coupling of the expressing cell into that network. Moreover, gap junctions appear to mediate interactions among homologous neurons that modulate process outgrowth and stability. We propose that the selective formation of GJs between developing neurons and perhaps glial cells in the CNS helps orchestrate not only cellular synaptic connectivity but also can have a pronounced effect on the arborization and morphology of those cells involved. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Fragger: a protein fragment picker for structural queries.

PubMed

Berenger, Francois; Simoncini, David; Voet, Arnout; Shrestha, Rojan; Zhang, Kam Y J

2017-01-01

Protein modeling and design activities often require querying the Protein Data Bank (PDB) with a structural fragment, possibly containing gaps. For some applications, it is preferable to work on a specific subset of the PDB or with unpublished structures. These requirements, along with specific user needs, motivated the creation of a new software to manage and query 3D protein fragments. Fragger is a protein fragment picker that allows protein fragment databases to be created and queried. All fragment lengths are supported and any set of PDB files can be used to create a database. Fragger can efficiently search a fragment database with a query fragment and a distance threshold. Matching fragments are ranked by distance to the query. The query fragment can have structural gaps and the allowed amino acid sequences matching a query can be constrained via a regular expression of one-letter amino acid codes. Fragger also incorporates a tool to compute the backbone RMSD of one versus many fragments in high throughput. Fragger should be useful for protein design, loop grafting and related structural bioinformatics tasks.

Endothelial connexin 32 regulates tissue factor expression induced by inflammatory stimulation and direct cell-cell interaction with activated cells.

PubMed

Okamoto, Takayuki; Akita, Nobuyuki; Hayashi, Tatsuya; Shimaoka, Motomu; Suzuki, Koji

2014-10-01

Endothelial cell (EC) interacts with adjacent EC through gap junction, and abnormal expression or function of Cxs is associated with cardiovascular diseases. In patients with endothelial dysfunction, the up-regulation of tissue factor (TF) expression promotes the pathogenic activation of blood coagulation, however the relationship between gap junctions and TF expression in ECs remains uncharacterized. ECs express the gap junction (GJ) proteins connexin32 (Cx32), Cx37, Cx40 and Cx43. We investigated the role of endothelial gap junctions, particularly Cx32, in modulating TF expression during vascular inflammation. Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor-α (TNF-α) and TF activity was assessed in the presence of GJ blockers and an inhibitory anti-Cx32 monoclonal antibody. Treatment with GJ blockers and anti-Cx32 monoclonal antibody enhanced the TNF-α-induced TF activity and mRNA expression in HUVECs. TNF-α-activated effector HUVECs or mouse MS-1 cells were co-cultured with non-stimulated acceptor HUVECs and TF expression in acceptor HUVECs was detected. Effector EC induced TF expression in adjacent acceptor HUVECs through direct cell-cell interaction. Cell-cell interaction induced TF expression was reduced by anti-intercellular adhesion molecule-1 (ICAM1) monoclonal antibody. Soluble ICAM1-Fc fusion protein promotes TF expression. GJ blockers and anti-Cx32 monoclonal antibody enhanced TF expression induced by cell-cell interaction and ICAM1-Fc treatment. Blockade of endothelial Cx32 increased TF expression induced by TNF-α stimulation and cell-cell interaction which was at least partly dependent upon ICAM1. These results suggest that direct Cx32-mediated interaction modulates TF expression in ECs during vascular inflammation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Rga6 is a fission yeast Rho GAP involved in Cdc42 regulation of polarized growth

PubMed Central

Revilla-Guarinos, M. T.; Martín-García, Rebeca; Villar-Tajadura, M. Antonia; Estravís, Miguel; Coll, Pedro M.; Pérez, Pilar

2016-01-01

Active Cdc42 is essential for the establishment of polarized growth. This GTPase is negatively regulated by the GTPase-activating proteins (GAPs), which are important for the spatial specificity of Cdc42 function. Rga4 is the only GAP described as negative regulator of fission yeast Cdc42. We report here that Rga6, another fission yeast Cdc42 GAP, shares some functions with Rga4. Cells lacking Rga6 are viable but slightly shorter and broader than wild type, and cells lacking Rga6 and Rga4 simultaneously are rounded. In these cells, active Cdc42 is observed all around the membrane. These additive effects indicate that both GAPs collaborate in the spatial regulation of active Cdc42. Rga6 localizes to the plasma membrane, forming clusters different from those formed by Rga4. A polybasic region at the Rga6 C-terminus is responsible for its membrane localization. Rga6-GFP fluorescence decreases considerably at the growing tips, and this decrease is dependent on the actin cables. Of note, in the absence of Rga6, the amplitude of active Cdc42 oscillations at the tips decreases, and less GTP-Cdc42 accumulates at the new end of the cells. We propose that Rga6 collaborates with Rga4 to spatially restrict active Cdc42 at the cell tips and maintain cell dimensions. PMID:26960792

Interactions of surface-displayed glycolytic enzymes of Mycoplasma pneumoniae with components of the human extracellular matrix.

PubMed

Gründel, Anne; Jacobs, Enno; Dumke, Roger

2016-12-01

Mycoplasma pneumoniae is a major cause of community-acquired respiratory infections worldwide. Due to the strongly reduced genome, the number of virulence factors expressed by this cell wall-less pathogen is limited. To further understand the processes during host colonization, we investigated the interactions of the previously confirmed surface-located glycolytic enzymes of M. pneumoniae (pyruvate dehydrogenase A-C [PdhA-C], glyceraldehyde-3-phosphate dehydrogenase [GapA], lactate dehydrogenase [Ldh], phosphoglycerate mutase [Pgm], pyruvate kinase [Pyk] and transketolase [Tkt]) to the human extracellular matrix (ECM) proteins fibrinogen (Fn), fibronectin (Fc), lactoferrin (Lf), laminin (Ln) and vitronectin (Vc), respectively. Concentration-dependent interactions between Fn and Vc and all eight recombinant proteins derived from glycolytic enzymes, between Ln and PdhB-C, GapA, Ldh, Pgm, Pyk and Tkt, between Lf and PdhA-C, GapA and Pyk, and between Fc and PdhC and GapA were demonstrated. In most cases, these associations are significantly influenced by ionic forces and by polyclonal sera against recombinant proteins. In immunoblotting, the complex of human plasminogen, activator (tissue-type or urokinase plasminogen activator) and glycolytic enzyme was not able to degrade Fc, Lf and Ln, respectively. In contrast, degradation of Vc was confirmed in the presence of all eight enzymes tested. Our data suggest that the multifaceted associations of surface-localized glycolytic enzymes play a potential role in the adhesion and invasion processes during infection of human respiratory mucosa by M. pneumoniae. Copyright © 2016 Elsevier GmbH. All rights reserved.

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation*

PubMed Central

Kistler, Samantha; George, Samuel D.; Kuhlmann, Nora; Garvey, Leslie; Huynh, Minh; Bagni, Rachel K.; Lammers, Michael; Der, Channing J.; Campbell, Sharon L.

2017-01-01

The KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cell-based properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity. PMID:28154176

GnRH Episodic Secretion Is Altered by Pharmacological Blockade of Gap Junctions: Possible Involvement of Glial Cells.

PubMed

Pinet-Charvet, Caroline; Geller, Sarah; Desroziers, Elodie; Ottogalli, Monique; Lomet, Didier; Georgelin, Christine; Tillet, Yves; Franceschini, Isabelle; Vaudin, Pascal; Duittoz, Anne

2016-01-01

Episodic release of GnRH is essential for reproductive function. In vitro studies have established that this episodic release is an endogenous property of GnRH neurons and that GnRH secretory pulses are associated with synchronization of GnRH neuron activity. The cellular mechanisms by which GnRH neurons synchronize remain largely unknown. There is no clear evidence of physical coupling of GnRH neurons through gap junctions to explain episodic synchronization. However, coupling of glial cells through gap junctions has been shown to regulate neuron activity in their microenvironment. The present study investigated whether glial cell communication through gap junctions plays a role in GnRH neuron activity and secretion in the mouse. Our findings show that Glial Fibrillary Acidic Protein-expressing glial cells located in the median eminence in close vicinity to GnRH fibers expressed Gja1 encoding connexin-43. To study the impact of glial-gap junction coupling on GnRH neuron activity, an in vitro model of primary cultures from mouse embryo nasal placodes was used. In this model, GnRH neurons possess a glial microenvironment and were able to release GnRH in an episodic manner. Our findings show that in vitro glial cells forming the microenvironment of GnRH neurons expressed connexin-43 and displayed functional gap junctions. Pharmacological blockade of the gap junctions with 50 μM 18-α-glycyrrhetinic acid decreased GnRH secretion by reducing pulse frequency and amplitude, suppressed neuronal synchronization and drastically reduced spontaneous electrical activity, all these effects were reversed upon 18-α-glycyrrhetinic acid washout.

An adaptor role for cytoplasmic Sam68 in modulating Src activity during cell polarization.

PubMed

Huot, Marc-Etienne; Brown, Claire M; Lamarche-Vane, Nathalie; Richard, Stéphane

2009-04-01

The Src-associated substrate during mitosis with a molecular mass of 68 kDa (Sam68) is predominantly nuclear and is known to associate with proteins containing the Src homology 3 (SH3) and SH2 domains. Although Sam68 is a Src substrate, little is known about the signaling pathway that link them. Src is known to be activated transiently after cell spreading, where it modulates the activity of small Rho GTPases. Herein we report that Sam68-deficient cells exhibit loss of cell polarity and cell migration. Interestingly, Sam68-deficient cells exhibited sustained Src activity after cell attachment, resulting in the constitutive tyrosine phosphorylation and activation of p190RhoGAP and its association with p120rasGAP. Consistently, we observed that Sam68-deficient cells exhibited deregulated RhoA and Rac1 activity. By using total internal reflection fluorescence microscopy, we observed Sam68 near the plasma membrane after cell attachment coinciding with phosphorylation of its C-terminal tyrosines and association with Csk. These findings show that Sam68 localizes near the plasma membrane during cell attachment and serves as an adaptor protein to modulate Src activity for proper signaling to small Rho GTPases.

The extracellular matrix controls gap junction protein expression and function in postnatal hippocampal neural progenitor cells

PubMed Central

Imbeault, Sophie; Gauvin, Lianne G; Toeg, Hadi D; Pettit, Alexandra; Sorbara, Catherine D; Migahed, Lamiaa; DesRoches, Rebecca; Menzies, A Sheila; Nishii, Kiyomasa; Paul, David L; Simon, Alexander M; Bennett, Steffany AL

2009-01-01

Background Gap junction protein and extracellular matrix signalling systems act in concert to influence developmental specification of neural stem and progenitor cells. It is not known how these two signalling systems interact. Here, we examined the role of ECM components in regulating connexin expression and function in postnatal hippocampal progenitor cells. Results We found that Cx26, Cx29, Cx30, Cx37, Cx40, Cx43, Cx45, and Cx47 mRNA and protein but only Cx32 and Cx36 mRNA are detected in distinct neural progenitor cell populations cultured in the absence of exogenous ECM. Multipotential Type 1 cells express Cx26, Cx30, and Cx43 protein. Their Type 2a progeny but not Type 2b and 3 neuronally committed progenitor cells additionally express Cx37, Cx40, and Cx45. Cx29 and Cx47 protein is detected in early oligodendrocyte progenitors and mature oligodendrocytes respectively. Engagement with a laminin substrate markedly increases Cx26 protein expression, decreases Cx40, Cx43, Cx45, and Cx47 protein expression, and alters subcellular localization of Cx30. These changes are associated with decreased neurogenesis. Further, laminin elicits the appearance of Cx32 protein in early oligodendrocyte progenitors and Cx36 protein in immature neurons. These changes impact upon functional connexin-mediated hemichannel activity but not gap junctional intercellular communication. Conclusion Together, these findings demonstrate a new role for extracellular matrix-cell interaction, specifically laminin, in the regulation of intrinsic connexin expression and function in postnatal neural progenitor cells. PMID:19236721

A Crosslinking Analysis of GAP-43 Interactions with Other Proteins in Differentiated N1E-115 Cells

PubMed Central

Ollom, Callise M.; Denny, John B.

2008-01-01

It has been suggested that GAP-43 (growth-associated protein) binds to various proteins in growing neurons as part of its mechanism of action. To test this hypothesis in vivo, differentiated N1E-115 neuroblastoma cells were labeled with [35S]-amino acids and were treated with a cleavable crosslinking reagent. The cells were lysed in detergent and the lysates were centrifuged at 100,000 × g to isolate crosslinked complexes. Following cleavage of the crosslinks and analysis by two-dimensional gel electrophoresis, it was found that the crosslinker increased the level of various proteins, and particularly actin, in this pellet fraction. However, GAP-43 was not present, suggesting that GAP-43 was not extensively crosslinked to proteins of the cytoskeleton and membrane skeleton and did not sediment with them. GAP-43 also did not sediment with the membrane skeleton following nonionic detergent lysis. Calmodulin, but not actin or other proposed interaction partners, co-immunoprecipitated with GAP-43 from the 100,000 × g supernatant following crosslinker addition to cells or cell lysates. Faint spots at 34 kDa and 60 kDa were also present. Additional GAP-43 was recovered from GAP-43 immunoprecipitation supernatants with anti-calmodulin but not with anti-actin. The results suggest that GAP-43 is not present in complexes with actin or other membrane skeletal or cytoskeletal proteins in these cells, but it is nevertheless possible that a small fraction of the total GAP-43 may interact with other proteins. PMID:19325830

The PriA Replication Restart Protein Blocks Replicase Access Prior to Helicase Assembly and Directs Template Specificity through Its ATPase Activity*

PubMed Central

Manhart, Carol M.; McHenry, Charles S.

2013-01-01

The PriA protein serves as an initiator for the restart of DNA replication on stalled replication forks and as a checkpoint protein that prevents the replicase from advancing in a strand displacement reaction on forks that do not contain a functional replicative helicase. We have developed a primosomal protein-dependent fluorescence resonance energy transfer (FRET) assay using a minimal fork substrate composed of synthetic oligonucleotides. We demonstrate that a self-loading reaction, which proceeds at high helicase concentrations, occurs by threading of a preassembled helicase over free 5′-ends, an event that can be blocked by attaching a steric block to the 5′-end or coating DNA with single-stranded DNA binding protein. The specificity of PriA for replication forks is regulated by its intrinsic ATPase. ATPase-defective PriA K230R shows a strong preference for substrates that contain no gap between the leading strand and the duplex portion of the fork, as demonstrated previously. Wild-type PriA prefers substrates with larger gaps, showing maximal activity on substrates on which PriA K230R is inactive. We demonstrate that PriA blocks replicase function on forks by blocking its binding. PMID:23264623

Inhibitors of connexin and pannexin channels as potential therapeutics

PubMed Central

Willebrords, Joost; Maes, Michaël; Crespo Yanguas, Sara; Vinken, Mathieu

2018-01-01

While gap junctions support the exchange of a number of molecules between neighboring cells, connexin hemichannels provide communication between the cytosol and the extracellular environment of an individual cell. The latter equally holds true for channels composed of pannexin proteins, which display an architecture reminiscent of connexin hemichannels. In physiological conditions, gap junctions are usually open, while connexin hemichannels and, to a lesser extent, pannexin channels are typically closed, yet they can be activated by a number of pathological triggers. Several agents are available to inhibit channels built up by connexin and pannexin proteins, including alcoholic substances, glycyrrhetinic acid, anesthetics and fatty acids. These compounds not always strictly distinguish between gap junctions, connexin hemichannels and pannexin channels, and may have effects on other targets as well. An exception lies with mimetic peptides, which reproduce specific amino acid sequences in connexin or pannexin primary protein structure. In this paper, a state-of-the-art overview is provided on inhibitors of cellular channels consisting of connexins and pannexins with specific focus on their mode-of-action and therapeutic potential. PMID:28720428

Capns1, a new binding partner of RasGAP-SH3 domain in K-Ras(V12) oncogenic cells: modulation of cell survival and migration.

PubMed

Pamonsinlapatham, Perayot; Gril, Brunilde; Dufour, Sylvie; Hadj-Slimane, Réda; Gigoux, Véronique; Pethe, Stéphanie; L'hoste, Sébastien; Camonis, Jacques; Garbay, Christiane; Raynaud, Françoise; Vidal, Michel

2008-11-01

Ras GTPase-activating protein (RasGAP) is hypothesized to be an effector of oncogenic Ras stimulating numerous downstream cellular signaling cascades involved in survival, proliferation and motility. In this study, we identified calpain small subunit-1 (Capns1) as a new RasGAP-SH3 domain binding partner, using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation assay and was found specific to cells expressing oncogenic K-Ras. We used confocal microscopy to analyze our stably transfected cell model producing mutant Ras (PC3Ras(V12)). Staining for RasGAP-SH3/Capns1 co-localization was two-fold stronger in the protrusions of Ras(V12) cells than in PC3 cells. RasGAP or Capns1 knockdown in PC3Ras(V12) cells induced a two- to three-fold increase in apoptosis. Capns1 gene silencing reduced the speed and increased the persistence of movement in PC3Ras(V12) cells. In contrast, RasGAP knockdown in PC3Ras(V12) cells increased cell migration. Knockdown of both proteins altered the speed and directionality of cell motility. Our findings suggest that RasGAP and Capns1 interaction in oncogenic Ras cells is involved in regulating migration and cell survival.

Development of Full Sweet, Umami, and Bitter Taste Responsiveness Requires Regulator of G protein Signaling-21 (RGS21).

PubMed

Schroer, Adam B; Gross, Joshua D; Kaski, Shane W; Wix, Kim; Siderovski, David P; Vandenbeuch, Aurelie; Setola, Vincent

2018-05-23

The mammalian tastes of sweet, umami, and bitter are initiated by activation of G protein-coupled receptors (GPCRs) of the T1R and T2R families on taste receptor cells. GPCRs signal via nucleotide exchange and hydrolysis, the latter hastened by GTPase-accelerating proteins (GAPs) that include the Regulators of G protein Signaling (RGS) protein family. We previously reported that RGS21, uniquely expressed in Type II taste receptor cells, decreases the potency of bitter-stimulated T2R signaling in cultured cells, consistent with its in vitro GAP activity. However, the role of RGS21 in organismal responses to GPCR-mediated tastants was not established. Here, we characterized mice lacking the Rgs21 fifth exon. Eliminating Rgs21 expression had no effect on body mass accumulation (a measure of alimentation), fungiform papillae number and morphology, circumvallate papillae morphology, and taste bud number. Two-bottle preference tests, however, revealed that Rgs21-null mice have blunted aversion to quinine and denatonium, and blunted preference for monosodium glutamate, the sweeteners sucrose and SC45647, and (surprisingly) NaCl. Observed reductions in GPCR-mediated tastant responses upon Rgs21 loss are opposite to original expectations, given that loss of RGS21-a GPCR signaling negative regulator-should lead to increased responsiveness to tastant-mediated GPCR signaling (all else being equal). Yet, reduced organismal tastant responses are consistent with observations of reduced chorda tympani nerve recordings in Rgs21-null mice. Reduced tastant-mediated responses and behaviors exhibited by adult mice lacking Rgs21 expression since birth have thus revealed an underappreciated requirement for a GPCR GAP to establish the full character of tastant signaling.

Ultrastructural demonstration of Cx43 gap junctions in induced pluripotent stem cells from human cord blood.

PubMed

Beckmann, Anja; Schubert, Madline; Hainz, Nadine; Haase, Alexandra; Martin, Ulrich; Tschernig, Thomas; Meier, Carola

2016-11-01

Gap junction proteins are essential for direct intercellular communication but also influence cellular differentiation and migration. The expression of various connexin gap junction proteins has been demonstrated in embryonic stem cells, with Cx43 being the most intensely studied. As Cx43 is the most prominent gap junction protein in the heart, cardiomyocyte-differentiated stem cells have been studied intensely. To date, however, little is known about the expression and the subcellular distribution of Cx43 in undifferentiated stem cells or about the structural arrangement of channels. We, therefore, here investigate expression of Cx43 in undifferentiated human cord-blood-derived induced pluripotent stem cells (hCBiPS2). For this purpose, we carried out quantitative real-time PCR and immunohistochemistry. For analysis of Cx43 ultrastructure and protein assembly, we performed freeze-fracture replica immunogold labeling (FRIL). Cx43 expression was detected at mRNA and protein level in hCBIPS2 cells. For the first time, ultrastructural data are presented on gap junction morphology in induced pluripotent stem (iPS) cells from cord blood: Our FRIL and electron microscopical analysis revealed the occurrence of gap junction plaques in undifferentiated iPS cells. In addition, these gap junctions were shown to contain the gap junction protein Cx43.

Photoperiod-Dependent Effects of 4-tert-Octylphenol on Adherens and Gap Junction Proteins in Bank Vole Seminiferous Tubules

PubMed Central

Kuras, Paulina; Lydka-Zarzycka, Marta; Bilinska, Barbara

2013-01-01

In the present study we evaluated in vivo and in vitro effects of 4-tert-octylphenol (OP) on the expression and distribution of adherens and gap junction proteins, N-cadherin, β-catenin, and connexin 43 (Cx43), in testes of seasonally breeding rodents, bank voles. We found that in bank vole testes expression and distribution of N-cadherin, β-catenin, and Cx43 were photoperiod dependent. Long-term treatment with OP (200 mg/kg b.w.) resulted in the reduction of junction proteins expressions (P < 0.05, P < 0.01) and their delocalization in the testes of males kept in long photoperiod, whereas in short-day animals slight increase of Cx43 (P < 0.05), N-cadherin, and β-catenin (statistically nonsignificant) levels was observed. Effects of OP appeared to be independent of FSH and were maintained during in vitro organ culture, indicating that OP acts directly on adherens and gap junction proteins in the testes. An experiment performed using an antiestrogen ICI 182,780 demonstrated that the biological effects of OP on β-catenin and Cx43 involve an estrogen receptor-mediated response. Taken together, in bank vole organization of adherens and gap junctions and their susceptibility to OP are related to the length of photoperiod. Alterations in cadherin/catenin and Cx43-based junction may partially result from activation of estrogen receptor α and/or β signaling pathway. PMID:23737770

2',5'-Dihydroxychalcone down-regulates endothelial connexin43 gap junctions and affects MAP kinase activation.

PubMed

Lee, Yi-Nan; Yeh, Hung-I; Tian, Tin-Yi; Lu, Wen-Wei; Ko, Yu-Shien; Tsai, Cheng-Ho

2002-09-30

We examined the effect of 2',5'-dihydroxychalcone on connexin43 (Cx43) expression and gap-junctional communication in human umbilical vein endothelial cells (HUVEC). The result showed that expression of Cx43 is rapidly reduced by 2',5'-dihydroxychalcone in a dose-dependent manner, Concomitantly, the communication function, determined by fluorescence recovery after photobleaching (FRAP), is decreased. We further investigated whether the mitogen-activated protein (MAP) kinase and the degradation pathway of gap junctions are involved in these processes. Although the change of Cx43 is not affected by the level of fetal calf serum (FCS) used in the medium, activation of MAP kinase varies, depending on the FCS level. At a low level (0.5%), the chalcone inhibits the activation, like PD98059, a specific inhibitor of MAP kinase kinase. However, at a high level (20%), MAP kinase is activated. On the other hand, the chalcone's down-regulating effect on Cx43, while is totally blocked by protease inhibitors leupeptin and N-acetyl-leucyl-norleucinal (ALLN), persists in the presence of PD98059, We concluded that 2',5'-dihydroxychalcone down-regulates Cx43 expression and gap-junctional communication in the HUVEC via enhancement of the proteolysis pathway, and this compound possesses dual effects on MAP kinase activation.

Impact of obesity on 7,12-dimethylbenz[a]anthracene-induced altered ovarian connexin gap junction proteins in female mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Nteeba, Jackson, E-mail: nteeba@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

The ovarian gap junction proteins alpha 4 (GJA4 or connexin 37; CX37), alpha 1 (GJA1 or connexin 43; CX43) and gamma 1 (GJC1 or connexin 45; CX45) are involved in cell communication and folliculogenesis. 7,12-dimethylbenz[a]anthracene (DMBA) alters Cx37 and Cx43 expression in cultured neonatal rat ovaries. Additionally, obesity has an additive effect on DMBA-induced ovarian cell death and follicle depletion, thus, we investigated in vivo impacts of obesity and DMBA on CX protein levels. Ovaries were collected from lean and obese mice aged 6, 12, 18, or 24 wks. A subset of 18 wk old mice (lean and obese) weremore » dosed with sesame oil or DMBA (1 mg/kg; ip) for 14 days and ovaries collected 3 days thereafter. Cx43 and Cx45 mRNA and protein levels decreased (P < 0.05) after 18 wks while Cx37 mRNA and protein levels decreased (P < 0.05) after 24 wks in obese ovaries. Cx37 mRNA and antral follicle protein staining intensity were reduced (P < 0.05) by obesity while total CX37 protein was reduced (P < 0.05) in DMBA exposed obese ovaries. Cx43 mRNA and total protein levels were decreased (P < 0.05) by DMBA in both lean and obese ovaries while basal protein staining intensity was reduced (P < 0.05) in obese controls. Cx45 mRNA, total protein and protein staining intensity level were decreased (P < 0.05) by obesity. These data support that obesity temporally alters gap junction protein expression and that DMBA-induced ovotoxicity may involve reduced gap junction protein function. - Highlights: • Ovarian gap junction proteins are affected by ovarian aging and obesity. • DMBA exposure negatively impacts gap junction proteins. • Altered gap junction proteins may contribute to infertility.« less

The General Amino Acid Permease FfGap1 of Fusarium fujikuroi Is Sorted to the Vacuole in a Nitrogen-Dependent, but Npr1 Kinase-Independent Manner

PubMed Central

Pfannmüller, Andreas; Wagner, Dominik; Sieber, Christian; Schönig, Birgit; Boeckstaens, Mélanie; Marini, Anna Maria; Tudzynski, Bettina

2015-01-01

The rice pathogenic fungus Fusarium fujikuroi is well known for the production of a broad spectrum of secondary metabolites (SMs) such as gibberellic acids (GAs), mycotoxins and pigments. The biosynthesis of most of these SMs strictly depends on nitrogen availability and of the activity of permeases of nitrogen sources, e.g. the ammonium and amino acid permeases. One of the three ammonium permeases, MepB, was recently shown to act not only as a transporter but also as a nitrogen sensor affecting the production of nitrogen-repressed SMs. Here we describe the identification of a general amino acid permease, FfGap1, among the 99 putative amino acid permeases (AAPs) in the genome of F. fujikuroi. FfGap1 is able to fully restore growth of the yeast gap1∆ mutant on several amino acids including citrulline and tryptophane. In S. cerevisiae, Gap1 activity is regulated by shuttling between the plasma membrane (nitrogen limiting conditions) and the vacuole (nitrogen sufficiency), which we also show for FfGap1. In yeast, the Npr1 serine/threonine kinase stabilizes the Gap1 position at the plasma membrane. Here, we identified and characterized three NPR1-homologous genes, encoding the putative protein kinases FfNpr1-1, FfNpr1-2 and FfNpr1-3 with significant similarity to yeast Npr1. Complementation of the yeast npr1Δ mutant with each of the three F. fujikuroi NPR1 homologues, resulted in partial restoration of ammonium, arginine and proline uptake by FfNPR1-1 while none of the three kinases affect growth on different nitrogen sources and nitrogen-dependent sorting of FfGap1 in F. fujikuroi. However, exchange of the putative ubiquitin-target lysine 9 (K9A) and 15 (K15A) residues of FfGap1 resulted in extended localization to the plasma membrane and increased protein stability independently of nitrogen availability. These data suggest a similar regulation of FfGap1 by nitrogen-dependent ubiquitination, but differences regarding the role of Fusarium Npr1 homologues compared to yeast. PMID:25909858

RPA and XPA interaction with DNA structures mimicking intermediates of the late stages in nucleotide excision repair

PubMed Central

Maltseva, Ekaterina A.

2018-01-01

Replication protein A (RPA) and the xeroderma pigmentosum group A (XPA) protein are indispensable for both pathways of nucleotide excision repair (NER). Here we analyze the interaction of RPA and XPA with DNA containing a flap and different size gaps that imitate intermediates of the late NER stages. Using gel mobility shift assays, we found that RPA affinity for DNA decreased when DNA contained both extended gap and similar sized flap in comparison with gapped-DNA structure. Moreover, crosslinking experiments with the flap-gap DNA revealed that RPA interacts mainly with the ssDNA platform within the long gap and contacts flap in DNA with a short gap. XPA exhibits higher affinity for bubble-DNA structures than to flap-gap-containing DNA. Protein titration analysis showed that formation of the RPA-XPA-DNA ternary complex depends on the protein concentration ratio and these proteins can function as independent players or in tandem. Using fluorescently-labelled RPA, direct interaction of this protein with XPA was detected and characterized quantitatively. The data obtained allow us to suggest that XPA can be involved in the post-incision NER stages via its interaction with RPA. PMID:29320546

RPA and XPA interaction with DNA structures mimicking intermediates of the late stages in nucleotide excision repair.

PubMed

Krasikova, Yuliya S; Rechkunova, Nadejda I; Maltseva, Ekaterina A; Lavrik, Olga I

2018-01-01

Replication protein A (RPA) and the xeroderma pigmentosum group A (XPA) protein are indispensable for both pathways of nucleotide excision repair (NER). Here we analyze the interaction of RPA and XPA with DNA containing a flap and different size gaps that imitate intermediates of the late NER stages. Using gel mobility shift assays, we found that RPA affinity for DNA decreased when DNA contained both extended gap and similar sized flap in comparison with gapped-DNA structure. Moreover, crosslinking experiments with the flap-gap DNA revealed that RPA interacts mainly with the ssDNA platform within the long gap and contacts flap in DNA with a short gap. XPA exhibits higher affinity for bubble-DNA structures than to flap-gap-containing DNA. Protein titration analysis showed that formation of the RPA-XPA-DNA ternary complex depends on the protein concentration ratio and these proteins can function as independent players or in tandem. Using fluorescently-labelled RPA, direct interaction of this protein with XPA was detected and characterized quantitatively. The data obtained allow us to suggest that XPA can be involved in the post-incision NER stages via its interaction with RPA.

Arf and Rho GAP adapter protein ARAP1 participates in the mobilization of TRAIL-R1/DR4 to the plasma membrane.

PubMed

Símová, Sárka; Klíma, Martin; Cermak, Lukas; Sourková, Vladimíra; Andera, Ladislav

2008-03-01

TRAIL, a ligand of the TNFalpha family, induces upon binding to its pro-death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 the apoptosis of cancer cells. Activated receptors incite the formation of the Death-Inducing Signaling Complex followed by the activation of the downstream apoptotic signaling. TRAIL-induced apoptosis is regulated at multiple levels, one of them being the presence and relative number of TRAIL pro- and anti-apoptotic receptors on the cytoplasmic membrane. In a yeast two-hybrid search for proteins that interact with the intracellular part (ICP) of DR4, we picked ARAP1, an adapter protein with ArfGAP and RhoGAP activities. In yeast, DR4(ICP) interacts with the alternatively spliced ARAP1 lacking 11 amino acids from the PH5 domain. Transfected ARAP1 co-precipitates with DR4 and co-localizes with it in the endoplasmic reticulum/Golgi, at the cytoplasmic membrane and in early endosomes of TRAIL-treated cells. ARAP1 knockdown significantly compromises the localization of DR4 at the cell surface of several tumor cell lines and slows down their TRAIL-induced death. ARAP1 overexpressed in HEL cells does not affect their TRAIL-induced apoptosis or the membrane localization of DR4, but it enhances the cell-surface presentation of phosphatidyl serine. Our data indicate that ARAP1 is likely involved in the regulation of the cell-specific trafficking of DR4 and might thus affect the efficacy of TRAIL-induced apoptosis.

Novel Gbeta Mimic Kelch Proteins Gpb1 and Gpb2 Connect G-Protein Signaling to Ras via Yeast Neurofibromin Homologs Ira 1 and Ira 2: A Model for Human NF1

DTIC Science & Technology

2006-03-01

Introduction Molecular switches composed of G-protein coupled receptors (GPCRs) and associated heterotrimeric G proteins transduce extracellular stimuli...We are investigating the molecular mechanisms by which the Ras GAP activity of the yeast neurofibromin homologs Ira1/2 is regulated as a model to...NF1 (For reviews, see Dasgupta and Gutmann, 2003; Parada, 2000; Zhu and Parada, 2002). It is therefore critical to elucidate the molecular mechanisms

Novel Gbeta Mimic Kelch Proteins (Gpb1 and Gpb2 Connect G-Protein Signaling to Ras via Yeast Neurofibromin Homologs Ira1 and Ira2. A Model for Human NF1

DTIC Science & Technology

2007-03-01

Saccharomyces cerevisiae and model fungus Cryptococcus neoformans as models to understand how the GAP activity of the yeast neurofibromin homologs, Ira1...another genetically tractable fungal model system, Cryptococcus neoformans, and identified two kelch repeat homologs that are involved in mating (Kem1 and...Kem2). To find kelch-repeat proteins involved in G protein signaling, Cryptococcus homologues of Gpb1/2, which interacts with and negatively

Altered cellular localization and hemichannel activities of KID syndrome associated connexin26 I30N and D50Y mutations.

PubMed

Aypek, Hande; Bay, Veysel; Meşe, Gülistan

2016-02-02

Gap junctions facilitate exchange of small molecules between adjacent cells, serving a crucial function for the maintenance of cellular homeostasis. Mutations in connexins, the basic unit of gap junctions, are associated with several human hereditary disorders. For example, mutations in connexin26 (Cx26) cause both non-syndromic deafness and syndromic deafness associated with skin abnormalities such as keratitis-ichthyosis-deafness (KID) syndrome. These mutations can alter the formation and function of gap junction channels through different mechanisms, and in turn interfere with various cellular processes leading to distinct disorders. The KID associated Cx26 mutations were mostly shown to result in elevated hemichannel activities. However, the effects of these aberrant hemichannels on cellular processes are recently being deciphered. Here, we assessed the effect of two Cx26 mutations associated with KID syndrome, Cx26I30N and D50Y, on protein biosynthesis and channel function in N2A and HeLa cells. Immunostaining experiments showed that Cx26I30N and D50Y failed to form gap junction plaques at cell-cell contact sites. Further, these mutations resulted in the retention of Cx26 protein in the Golgi apparatus. Examination of hemichannel function by fluorescent dye uptake assays revealed that cells with Cx26I30N and D50Y mutations had increased dye uptake compared to Cx26WT (wild-type) containing cells, indicating abnormal hemichannel activities. Cells with mutant proteins had elevated intracellular calcium levels compared to Cx26WT transfected cells, which were abolished by a hemichannel blocker, carbenoxolone (CBX), as measured by Fluo-3 AM loading and flow cytometry. Here, we demonstrated that Cx26I30N and D50Y mutations resulted in the formation of aberrant hemichannels that might result in elevated intracellular calcium levels, a process which may contribute to the hyperproliferative epidermal phenotypes of KID syndrome.

R4 RGS Proteins: Regulation of G Protein Signaling and Beyond

PubMed Central

Bansal, Geetanjali; Druey, Kirk M.; Xie, Zhihui

2007-01-01

The Regulators of G protein Signaling (RGS) proteins were initially characterized as inhibitors of signal transduction cascades initiated by G-protein-coupled receptors (GPCRs) because of their ability to increase the intrinsic GTPase activity of heterotrimeric G proteins. This GTPase accelerating (GAP) activity enhances G protein deactivation and promotes desensitization. However, in addition to this signature trait, emerging data have revealed an expanding network of proteins, lipids, and ions that interact with RGS proteins and confer additional regulatory functions. This review highlights recent advances in our understanding of the physiological functions of one subfamily of RGS proteins with a high degree of homology (B/R4) gleaned from recent studies of knockout mice or cells with reduced RGS expression. We also discuss some of the newly-appreciated interactions of RGS proteins with cellular factors that suggest RGS control of several components of G-protein-mediated pathways as well as a diverse array of non-GPCR-mediated biological responses. PMID:18006065

LRP6 acts as a scaffold protein in cardiac gap junction assembly

PubMed Central

Li, Jun; Li, Changming; Liang, Dandan; Lv, Fei; Yuan, Tianyou; The, Erlinda; Ma, Xiue; Wu, Yahan; Zhen, Lixiao; Xie, Duanyang; Wang, Shiyi; Liu, Yuan; Huang, Jian; Shi, Jingyi; Liu, Yi; Shi, Dan; Xu, Liang; Lin, Li; Peng, Luying; Cui, Jianmin; Zhu, Weidong; Chen, Yi-Han

2016-01-01

Low-density lipoprotein receptor-related protein 6 (LRP6) is a Wnt co-receptor in the canonical Wnt/β-catenin signalling. Here, we report the scaffold function of LRP6 in gap junction formation of cardiomyocytes. Cardiac LRP6 is spatially restricted to intercalated discs and binds to gap junction protein connexin 43 (Cx43). A deficiency in LRP6 disrupts Cx43 gap junction formation and thereby impairs the cell-to-cell coupling, which is independent of Wnt/β-catenin signalling. The defect in Cx43 gap junction resulting from LRP6 reduction is attributable to the defective traffic of de novo Cx43 proteins from the endoplasmic reticulum to the Golgi apparatus, leading to the lysosomal degradation of Cx43 proteins. Accordingly, the hearts of conditional cardiac-specific Lrp6-knockout mice consistently exhibit overt reduction of Cx43 gap junction plaques without any abnormality in Wnt signalling and are predisposed to lethal arrhythmias. These findings uncover a distinct role of LRP6 as a platform for intracellular protein trafficking. PMID:27250245

Conservation of Three-Dimensional Helix-Loop-Helix Structure through the Vertebrate Lineage Reopens the Cold Case of Gonadotropin-Releasing Hormone-Associated Peptide.

PubMed

Pérez Sirkin, Daniela I; Lafont, Anne-Gaëlle; Kamech, Nédia; Somoza, Gustavo M; Vissio, Paula G; Dufour, Sylvie

2017-01-01

GnRH-associated peptide (GAP) is the C-terminal portion of the gonadotropin-releasing hormone (GnRH) preprohormone. Although it was reported in mammals that GAP may act as a prolactin-inhibiting factor and can be co-secreted with GnRH into the hypophyseal portal blood, GAP has been practically out of the research circuit for about 20 years. Comparative studies highlighted the low conservation of GAP primary amino acid sequences among vertebrates, contributing to consider that this peptide only participates in the folding or carrying process of GnRH. Considering that the three-dimensional (3D) structure of a protein may define its function, the aim of this study was to evaluate if GAP sequences and 3D structures are conserved in the vertebrate lineage. GAP sequences from various vertebrates were retrieved from databases. Analysis of primary amino acid sequence identity and similarity, molecular phylogeny, and prediction of 3D structures were performed. Amino acid sequence comparison and phylogeny analyses confirmed the large variation of GAP sequences throughout vertebrate radiation. In contrast, prediction of the 3D structure revealed a striking conservation of the 3D structure of GAP1 (GAP associated with the hypophysiotropic type 1 GnRH), despite low amino acid sequence conservation. This GAP1 peptide presented a typical helix-loop-helix (HLH) structure in all the vertebrate species analyzed. This HLH structure could also be predicted for GAP2 in some but not all vertebrate species and in none of the GAP3 analyzed. These results allowed us to infer that selective pressures have maintained GAP1 HLH structure throughout the vertebrate lineage. The conservation of the HLH motif, known to confer biological activity to various proteins, suggests that GAP1 peptides may exert some hypophysiotropic biological functions across vertebrate radiation.

Conservation of Three-Dimensional Helix-Loop-Helix Structure through the Vertebrate Lineage Reopens the Cold Case of Gonadotropin-Releasing Hormone-Associated Peptide

PubMed Central

Pérez Sirkin, Daniela I.; Lafont, Anne-Gaëlle; Kamech, Nédia; Somoza, Gustavo M.; Vissio, Paula G.; Dufour, Sylvie

2017-01-01

GnRH-associated peptide (GAP) is the C-terminal portion of the gonadotropin-releasing hormone (GnRH) preprohormone. Although it was reported in mammals that GAP may act as a prolactin-inhibiting factor and can be co-secreted with GnRH into the hypophyseal portal blood, GAP has been practically out of the research circuit for about 20 years. Comparative studies highlighted the low conservation of GAP primary amino acid sequences among vertebrates, contributing to consider that this peptide only participates in the folding or carrying process of GnRH. Considering that the three-dimensional (3D) structure of a protein may define its function, the aim of this study was to evaluate if GAP sequences and 3D structures are conserved in the vertebrate lineage. GAP sequences from various vertebrates were retrieved from databases. Analysis of primary amino acid sequence identity and similarity, molecular phylogeny, and prediction of 3D structures were performed. Amino acid sequence comparison and phylogeny analyses confirmed the large variation of GAP sequences throughout vertebrate radiation. In contrast, prediction of the 3D structure revealed a striking conservation of the 3D structure of GAP1 (GAP associated with the hypophysiotropic type 1 GnRH), despite low amino acid sequence conservation. This GAP1 peptide presented a typical helix-loop-helix (HLH) structure in all the vertebrate species analyzed. This HLH structure could also be predicted for GAP2 in some but not all vertebrate species and in none of the GAP3 analyzed. These results allowed us to infer that selective pressures have maintained GAP1 HLH structure throughout the vertebrate lineage. The conservation of the HLH motif, known to confer biological activity to various proteins, suggests that GAP1 peptides may exert some hypophysiotropic biological functions across vertebrate radiation. PMID:28878737

Structural Basis for TSC-1 TSC-2 Complex Formation

DTIC Science & Technology

2008-03-01

mental retardation and autism . Brain lesions include cerebral cortical tubers, subependymal nodules, and retinal hamartomas. Patients often develop...Rheb) (6,7). Rheb activates the mammalian target of Rapamycin ( mTOR ) and inactivates B-Raf (7). Activation of mTOR leads to increased protein...activity and activation of mTOR . Several unrelated patients with tuberous sclerosis have point mutations in the TSC2 GAP domain that influence the

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation.

PubMed

Yin, Guowei; Kistler, Samantha; George, Samuel D; Kuhlmann, Nora; Garvey, Leslie; Huynh, Minh; Bagni, Rachel K; Lammers, Michael; Der, Channing J; Campbell, Sharon L

2017-03-17

The KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cell-based properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional role of connexin43 gap junction channels in adult mouse heart assessed by inducible gene deletion.

PubMed

Eckardt, D; Theis, M; Degen, J; Ott, T; van Rijen, H V M; Kirchhoff, S; Kim, J-S; de Bakker, J M T; Willecke, K

2004-01-01

The gap junction protein Connexin43 (Cx43) is expressed in various cell types during embryonic development and in adult mice. Cx43 null mice (Cx43-/-) die perinatally due to cardiac malformation. In order to define the major functional role of Cx43 gap junction channels in adult mice and to circumvent perinatal death as well as direct or indirect compensation of Cx43 deficiency during development, we established a novel conditional Cx43 mouse mutant. To ablate Cx43 in adult mice in all cells that express Cx43 at a certain time, we targeted the 4-hydroxytamoxifen inducible Cre recombinase, Cre-ER(T), into the endogenous Cx43 locus. This approach left only one Cx43 coding region to be deleted upon induction of Cre-ER(T) activity. Highly efficient inducible ablation of Cx43 was shown in an embryonic stem cell test system and in adult mice. Although Cx43 protein was decreased in different tissues after induction of Cre-ER(T)-mediated recombination, cardiac abnormalities most likely account for death of those mice. Surface and telemetric ECG recordings revealed significant delay of ventricular activation and death during periods of bradyarrhythmia preceded by tachycardias. This novel approach of inducible ablation of Cx43 highlights the functional importance of normal activation of ventricular cardiomyocytes mediated by Cx43 gap junction channels in adult mouse heart to prevent initiation of fatal arrhythmias. The new mouse model should be useful for further analyses of molecular changes initiated by acute loss of Cx43 expression in various cell types.

RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development

PubMed Central

Martin-Vilchez, Samuel; Whitmore, Leanna; Asmussen, Hannelore; Zareno, Jessica; Horwitz, Rick; Newell-Litwa, Karen

2017-01-01

Small RhoGTPases regulate changes in post-synaptic spine morphology and density that support learning and memory. They are also major targets of synaptic disorders, including Autism. Here we sought to determine whether upstream RhoGTPase regulators, including GEFs, GAPs, and GDIs, sculpt specific stages of synaptic development. The majority of examined molecules uniquely regulate either early spine precursor formation or later maturation. Specifically, an activator of actin polymerization, the Rac1 GEF β-PIX, drives spine precursor formation, whereas both FRABIN, a Cdc42 GEF, and OLIGOPHRENIN-1, a RhoA GAP, regulate spine precursor elongation. However, in later development, a novel Rac1 GAP, ARHGAP23, and RhoGDIs inactivate actomyosin dynamics to stabilize mature synapses. Our observations demonstrate that specific combinations of RhoGTPase regulatory proteins temporally balance RhoGTPase activity during post-synaptic spine development. PMID:28114311

RanGAP1 is a continuous marker of the Arabidopsis cell division plane

PubMed Central

Xu, Xianfeng Morgan; Zhao, Qiao; Rodrigo-Peiris, Thushani; Brkljacic, Jelena; He, Chao Sylvia; Müller, Sabine; Meier, Iris

2008-01-01

In higher plants, the plane of cell division is faithfully predicted by the preprophase band (PPB). The PPB, a cortical ring of microtubules and F-actin, disassembles upon nuclear-envelope breakdown. During cytokinesis, the expanding cell plate fuses with the plasma membrane at the cortical division site, the site of the former PPB. The nature of the “molecular memory” that is left behind by the PPB and is proposed to guide the cell plate to the cortical division site is unknown. RanGAP is the GTPase activating protein of the small GTPase Ran, which provides spatial information for nucleocytoplasmic transport and various mitotic processes in animals. Here, we show that, in dividing root cells, Arabidopsis RanGAP1 concentrates at the PPB and remains associated with the cortical division site during mitosis and cytokinesis, requiring its N-terminal targeting domain. In a fass/ton2 mutant, which affects PPB formation, RanGAP1 recruitment to the PPB site is lost, while its PPB retention is microtubule-independent. RanGAP1 persistence at the cortical division site, but not its initial accumulation at the PPB requires the 2 cytokinesis-regulating kinesins POK1 and POK2. Depletion of RanGAP by inducible RNAi leads to oblique cell walls and cell-wall stubs in root cell files, consistent with cytokinesis defects. We propose that Arabidopsis RanGAP, a continuous positive protein marker of the plant division plane, has a role in spatial signaling during plant cell division. PMID:19011093

Inhibition of gap-junctional intercellular communication and activation of mitogen-activated protein kinases by cyanobacterial extracts--indications of novel tumor-promoting cyanotoxins?

PubMed

Bláha, Ludĕk; Babica, Pavel; Hilscherová, Klára; Upham, Brad L

2010-01-01

Toxicity and liver tumor promotion of cyanotoxins microcystins have been extensively studied. However, recent studies document that other metabolites present in the complex cyanobacterial water blooms may also have adverse health effects. In this study we used rat liver epithelial stem-like cells (WB-F344) to examine the effects of cyanobacterial extracts on two established markers of tumor promotion, inhibition of gap-junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) - ERK1/2. Extracts of cyanobacteria (laboratory cultures of Microcystis aeruginosa and Aphanizomenon flos-aquae and water blooms dominated by these species) inhibited GJIC and activated MAPKs in a dose-dependent manner (effective concentrations ranging 0.5-5mgd.w./mL). Effects were independent of the microcystin content and the strongest responses were elicited by the extracts of Aphanizomenon sp. Neither pure microcystin-LR nor cylindrospermopsin inhibited GJIC or activated MAPKs. Modulations of GJIC and MAPKs appeared to be specific to cyanobacterial extracts since extracts from green alga Chlamydomonas reinhardtii, heterotrophic bacterium Klebsiella terrigena, and isolated bacterial lipopolysaccharides had no comparable effects. Our study provides the first evidence on the existence of unknown cyanobacterial toxic metabolites that affect in vitro biomarkers of tumor promotion, i.e. inhibition of GJIC and activation of MAPKs.

Interaction of a Blumeria graminis f. sp. hordei effector candidate with a barley ARF-GAP suggests that host vesicle trafficking is a fungal pathogenicity target.

PubMed

Schmidt, Sarah M; Kuhn, Hannah; Micali, Cristina; Liller, Corinna; Kwaaitaal, Mark; Panstruga, Ralph

2014-08-01

Filamentous phytopathogens, such as fungi and oomycetes, secrete effector proteins to establish successful interactions with their plant hosts. In contrast with oomycetes, little is known about effector functions in true fungi. We used a bioinformatics pipeline to identify Blumeria effector candidates (BECs) from the obligate biotrophic barley powdery mildew pathogen, Blumeria graminis f. sp. hordei (Bgh). BEC1-BEC5 are expressed at different time points during barley infection. BEC1, BEC2 and BEC4 have orthologues in the Arabidopsis thaliana-infecting powdery mildew fungus Golovinomyces orontii. Arabidopsis lines stably expressing the G. orontii BEC2 orthologue, GoEC2, are more susceptible to infection with the non-adapted fungus Erysiphe pisi, suggesting that GoEC2 contributes to powdery mildew virulence. For BEC3 and BEC4, we identified thiopurine methyltransferase, a ubiquitin-conjugating enzyme, and an ADP ribosylation factor-GTPase-activating protein (ARF-GAP) as potential host targets. Arabidopsis knockout lines of the respective HvARF-GAP orthologue (AtAGD5) allowed higher entry levels of E. pisi, but exhibited elevated resistance to the oomycete Hyaloperonospora arabidopsidis. We hypothesize that ARF-GAP proteins are conserved targets of powdery and downy mildew effectors, and we speculate that BEC4 might interfere with defence-associated host vesicle trafficking. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Phosphorylation of Rga2, a Cdc42 GAP, by CDK/Hgc1 is crucial for Candida albicans hyphal growth

PubMed Central

Zheng, Xin-De; Lee, Raymond Teck Ho; Wang, Yan-Ming; Lin, Qi-Shan; Wang, Yue

2007-01-01

Cyclin-dependent kinases (CDKs) control yeast morphogenesis, although how they regulate the polarity machinery remains unclear. The dimorphic fungus Candida albicans uses Cdc28/Hgc1, a CDK/cyclin complex, to promote persistent actin polarization for hyphal growth. Here, we report that Rga2, a GTPase-activating protein (GAP) of the central polarity regulator Cdc42, undergoes Hgc1-dependent hyperphosphorylation. Using the analog-sensitive Cdc28as mutant, we confirmed that Cdc28 controls Rga2 phosphorylation in vitro and in vivo. Deleting RGA2 produced elongated yeast cells without apparent effect on hyphal morphogenesis. However, deleting it or inactivating its GAP activity restored hyphal growth in hgc1Δ mutants, suggesting that Rga2 represses hyphal development and Cdc28/Hgc1 inactivates it upon hyphal induction. We provide evidence that Cdc28/Hgc1 may act to prevent Rga2 from localizing to hyphal tips, leading to localized Cdc42 activation for hyphal extension. Rga2 also undergoes transient Cdc28-dependent hyperphosphorylation at bud emergence, suggesting that regulating a GAP(s) of Cdc42 by CDKs may play an important role in governing different forms of polarized morphogenesis in yeast. This study reveals a direct molecular link between CDKs and the polarity machinery. PMID:17673907

The gap junction channel protein connexin 43 is covalently modified and regulated by SUMOylation.

PubMed

Kjenseth, Ane; Fykerud, Tone A; Sirnes, Solveig; Bruun, Jarle; Yohannes, Zeremariam; Kolberg, Matthias; Omori, Yasufumi; Rivedal, Edgar; Leithe, Edward

2012-05-04

SUMOylation is a posttranslational modification in which a member of the small ubiquitin-like modifier (SUMO) family of proteins is conjugated to lysine residues in specific target proteins. Most known SUMOylation target proteins are located in the nucleus, but there is increasing evidence that SUMO may also be a key determinant of many extranuclear processes. Gap junctions consist of arrays of intercellular channels that provide direct transfer of ions and small molecules between adjacent cells. Gap junction channels are formed by integral membrane proteins called connexins, of which the best-studied isoform is connexin 43 (Cx43). Here we show that Cx43 is posttranslationally modified by SUMOylation. The data suggest that the SUMO system regulates the Cx43 protein level and the level of functional Cx43 gap junctions at the plasma membrane. Cx43 was found to be modified by SUMO-1, -2, and -3. Evidence is provided that the membrane-proximal lysines at positions 144 and 237, located in the Cx43 intracellular loop and C-terminal tail, respectively, act as SUMO conjugation sites. Mutations of lysine 144 or lysine 237 resulted in reduced Cx43 SUMOylation and reduced Cx43 protein and gap junction levels. Altogether, these data identify Cx43 as a SUMOylation target protein and represent the first evidence that gap junctions are regulated by the SUMO system.

Regulating the Regulator: Post-Translational Modification of Ras

PubMed Central

Ahearn, Ian M.; Haigis, Kevin; Bar-Sagi, Dafna; Philips, Mark R.

2013-01-01

Ras proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on Ras is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which regulate the activation state of Ras without covalently modifying it. In contrast, post-translational modifications (PTMs) of Ras proteins direct them to various cellular membranes and, in some cases, modulate GTP–GDP exchange. Important Ras PTMs include the constitutive and irreversible remodelling of its C-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications including phosphorylation, peptidyl-proly isomerisation, mono- and di-ubiquitination, nitrosylation, ADP ribosylation and glucosylation. PMID:22189424

Nck-Interacting Ste20 Kinase Couples Eph Receptors to c-Jun N-Terminal Kinase and Integrin Activation

PubMed Central

Becker, Elena; Huynh-Do, Uyen; Holland, Sacha; Pawson, Tony; Daniel, Tom O.; Skolnik, Edward Y.

2000-01-01

The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62dok, RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62dok most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs. PMID:10669731

Mitogenic signals and transforming potential of Nyk, a newly identified neural cell adhesion molecule-related receptor tyrosine kinase.

PubMed Central

Ling, L; Kung, H J

1995-01-01

Nyk/Mer is a recently identified receptor tyrosine kinase with neural cell adhesion molecule-like structure (two immunoglobulin G-like domains and two fibronectin III-like domains) in its extracellular region and belongs to the Ufo/Axl family of receptors. The ligand for Nyk/Mer is presently unknown, as are the signal transduction pathways mediated by this receptor. We constructed and expressed a chimeric receptor (Fms-Nyk) composed of the extracellular domain of the human colony-stimulating factor 1 receptor (Fms) and the transmembrane and cytoplasmic domains of human Nyk/Mer in NIH 3T3 fibroblasts in order to investigate the mitogenic signaling and biochemical properties of Nyk/Mer. Colony-stimulating factor 1 stimulation of the Fms-Nyk chimeric receptor in transfected NIH 3T3 fibroblasts leads to a transformed phenotype and generates a proliferative response in the absence of other growth factors. We show that phospholipase C gamma, phosphatidylinositol 3-kinase/p70 S6 kinase, Shc, Grb2, Raf-1, and mitogen-activated protein kinase are downstream components of the Nyk/Mer signal transduction pathways. In addition, Nyk/Mer weakly activates p90rsk, while stress-activated protein kinase, Ras GTPase-activating protein (GAP), and GAP-associated p62 and p190 proteins are not activated or tyrosine phosphorylated by Nyk/Mer. An analysis comparing the Nyk/Mer signal cascade with that of the epidermal growth factor receptor indicates substrate preferences by these two receptors. Our results provide a detailed description of the Nyk/Mer signaling pathways. Given the structural similarity between the Ufo/Axl family receptors, some of the information may also be applied to other members of this receptor tyrosine kinase family. PMID:8524223

Docosahexaenoic acid induces ERK1/2 activation and neuritogenesis via intracellular reactive oxygen species production in human neuroblastoma SH-SY5Y cells.

PubMed

Wu, Haitao; Ichikawa, Sanae; Tani, Chiharu; Zhu, Beiwei; Tada, Mikiro; Shimoishi, Yasuaki; Murata, Yoshiyuki; Nakamura, Yoshimasa

2009-01-01

Docosahexaenoic acid (22: 6n-3; DHA) is a long chain polyunsaturated fatty acid that exists highly enriched in fish oil, and it is one of the low molecular weight food chemicals which can pass a blood brain barrier. A preliminary survey of several fatty acids for expression of growth-associated protein-43 (GAP-43), a marker of axonal growth, identified DHA as one of the most potent inducers. The human neuroblastoma SH-SY5Y cells exposed to DHA showed significant and dose-dependent increases in the percentage of cells with longer neurites. To elucidate signaling mechanisms involved in DHA-enhanced basal neuritogenesis, we examined the role of extracellular signal-regulated kinase (ERK)1/2 and intracellular reactive oxygen species (ROS) production using SH-SY5Y cells. From immunoblotting experiments, we observed that DHA induced the ROS production, protein tyrosine phosphatase inhibition, mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) phosphorylation, and sequentially ERK1/2 phosphorylation, the last of which was significantly reduced by MEK inhibitor U0126. Both antioxidants and MEK inhibitor affected DHA-induced GAP-43 expression, whereas the specific PI3K inhibitor LY294002 did not. We found that total protein tyrosine phosphatase activity was also downregulated by DHA treatment, which was counteracted by antioxidant pretreatment. These results suggest that the ROS-dependent ERK pathway, rather than PI3K, plays an important role during DHA-enhanced neurite outgrowth.

Tracking Glideosome-associated protein 50 reveals the development and organization of the inner membrane complex of Plasmodium falciparum.

PubMed

Yeoman, Jeffrey A; Hanssen, Eric; Maier, Alexander G; Klonis, Nectarios; Maco, Bohumil; Baum, Jake; Turnbull, Lynne; Whitchurch, Cynthia B; Dixon, Matthew W A; Tilley, Leann

2011-04-01

The most deadly of the human malaria parasites, Plasmodium falciparum, has different stages specialized for invasion of hepatocytes, erythrocytes, and the mosquito gut wall. In each case, host cell invasion is powered by an actin-myosin motor complex that is linked to an inner membrane complex (IMC) via a membrane anchor called the glideosome-associated protein 50 (PfGAP50). We generated P. falciparum transfectants expressing green fluorescent protein (GFP) chimeras of PfGAP50 (PfGAP50-GFP). Using immunoprecipitation and fluorescence photobleaching, we show that C-terminally tagged PfGAP50-GFP can form a complex with endogenous copies of the linker protein PfGAP45 and the myosin A tail domain-interacting protein (MTIP). Full-length PfGAP50-GFP is located in the endoplasmic reticulum in early-stage parasites and then redistributes to apical caps during the formation of daughter merozoites. In the final stage of schizogony, the PfGAP50-GFP profile extends further around the merozoite surface. Three-dimensional (3D) structured illumination microscopy reveals the early-stage IMC as a doubly punctured flat ellipsoid that separates to form claw-shaped apposed structures. A GFP fusion of PfGAP50 lacking the C-terminal membrane anchor is misdirected to the parasitophorous vacuole. Replacement of the acid phosphatase homology domain of PfGAP50 with GFP appears to allow correct trafficking of the chimera but confers a growth disadvantage.

PI3K/Akt signaling is involved in the disruption of gap junctional communication caused by v-Src and TNF-α.

PubMed

Ito, Satoko; Hyodo, Toshinori; Hasegawa, Hitoki; Yuan, Hong; Hamaguchi, Michinari; Senga, Takeshi

2010-09-17

Gap junctional communication, which is mediated by the connexin protein family, is essential for the maintenance of normal tissue function and homeostasis. Loss of intercellular communication results in a failure to coordinately regulate cellular functions, and it can facilitate tumorigenesis. Expression of oncogenes and stimulation with cytokines has been shown to suppress intercellular communication; however, the exact mechanism by which intercellular communication is disrupted by these factors remains uncertain. In this report, we show that Akt is essential for the disruption of gap junctional communication in v-Src-transformed cells. In addition, inhibition of Akt restores gap junctional communication after it is suppressed by TNF-α signaling. Furthermore, we demonstrate that the expression of a constitutively active form of Akt1, but not of Akt2 or Akt3, is sufficient to suppress gap junctional communication. Our results clearly define Akt1 as one of the critical regulators of gap junctional communication. Copyright © 2010 Elsevier Inc. All rights reserved.

RAB-5 and RAB-10 cooperate to regulate neuropeptide release in Caenorhabditis elegans

PubMed Central

Sasidharan, Nikhil; Sumakovic, Marija; Hannemann, Mandy; Hegermann, Jan; Liewald, Jana F.; Olendrowitz, Christian; Koenig, Sabine; Grant, Barth D.; Rizzoli, Silvio O.; Gottschalk, Alexander; Eimer, Stefan

2012-01-01

Neurons secrete neuropeptides from dense core vesicles (DCVs) to modulate neuronal activity. Little is known about how neurons manage to differentially regulate the release of synaptic vesicles (SVs) and DCVs. To analyze this, we screened all Caenorhabditis elegans Rab GTPases and Tre2/Bub2/Cdc16 (TBC) domain containing GTPase-activating proteins (GAPs) for defects in DCV release from C. elegans motoneurons. rab-5 and rab-10 mutants show severe defects in DCV secretion, whereas SV exocytosis is unaffected. We identified TBC-2 and TBC-4 as putative GAPs for RAB-5 and RAB-10, respectively. Multiple Rabs and RabGAPs are typically organized in cascades that confer directionality to membrane-trafficking processes. We show here that the formation of release-competent DCVs requires a reciprocal exclusion cascade coupling RAB-5 and RAB-10, in which each of the two Rabs recruits the other’s GAP molecule. This contributes to a separation of RAB-5 and RAB-10 domains at the Golgi–endosomal interface, which is lost when either of the two GAPs is inactivated. Taken together, our data suggest that RAB-5 and RAB-10 cooperate to locally exclude each other at an essential stage during DCV sorting. PMID:23100538

Mechanisms of CDC-42 activation during contact-induced cell polarization.

PubMed

Chan, Emily; Nance, Jeremy

2013-04-01

Polarization of early embryos provides a foundation to execute essential patterning and morphogenetic events. In Caenorhabditis elegans, cell contacts polarize early embryos along their radial axis by excluding the cortical polarity protein PAR-6 from sites of cell contact, thereby restricting PAR-6 to contact-free cell surfaces. Radial polarization requires the cortically enriched Rho GTPase CDC-42, which in its active form recruits PAR-6 through direct binding. The Rho GTPase activating protein (RhoGAP) PAC-1, which localizes specifically to cell contacts, triggers radial polarization by inactivating CDC-42 at these sites. The mechanisms responsible for activating CDC-42 at contact-free surfaces are unknown. Here, in an overexpression screen of Rho guanine nucleotide exchange factors (RhoGEFs), which can activate Rho GTPases, we identify CGEF-1 and ECT-2 as RhoGEFs that act through CDC-42 to recruit PAR-6 to the cortex. We show that ECT-2 and CGEF-1 localize to the cell surface and that removing their activity causes a reduction in levels of cortical PAR-6. Through a structure-function analysis, we show that the tandem DH-PH domains of CGEF-1 and ECT-2 are sufficient for GEF activity, but that regions outside of these domains target each protein to the cell surface. Finally, we provide evidence suggesting that the N-terminal region of ECT-2 may direct its in vivo preference for CDC-42 over another known target, the Rho GTPase RHO-1. We propose that radial polarization results from a competition between RhoGEFs, which activate CDC-42 throughout the cortex, and the RhoGAP PAC-1, which inactivates CDC-42 at cell contacts.

Mechanisms of CDC-42 activation during contact-induced cell polarization

PubMed Central

Chan, Emily; Nance, Jeremy

2013-01-01

Summary Polarization of early embryos provides a foundation to execute essential patterning and morphogenetic events. In Caenorhabditis elegans, cell contacts polarize early embryos along their radial axis by excluding the cortical polarity protein PAR-6 from sites of cell contact, thereby restricting PAR-6 to contact-free cell surfaces. Radial polarization requires the cortically enriched Rho GTPase CDC-42, which in its active form recruits PAR-6 through direct binding. The Rho GTPase activating protein (RhoGAP) PAC-1, which localizes specifically to cell contacts, triggers radial polarization by inactivating CDC-42 at these sites. The mechanisms responsible for activating CDC-42 at contact-free surfaces are unknown. Here, in an overexpression screen of Rho guanine nucleotide exchange factors (RhoGEFs), which can activate Rho GTPases, we identify CGEF-1 and ECT-2 as RhoGEFs that act through CDC-42 to recruit PAR-6 to the cortex. We show that ECT-2 and CGEF-1 localize to the cell surface and that removing their activity causes a reduction in levels of cortical PAR-6. Through a structure–function analysis, we show that the tandem DH-PH domains of CGEF-1 and ECT-2 are sufficient for GEF activity, but that regions outside of these domains target each protein to the cell surface. Finally, we provide evidence suggesting that the N-terminal region of ECT-2 may direct its in vivo preference for CDC-42 over another known target, the Rho GTPase RHO-1. We propose that radial polarization results from a competition between RhoGEFs, which activate CDC-42 throughout the cortex, and the RhoGAP PAC-1, which inactivates CDC-42 at cell contacts. PMID:23424200

Machineries regulating the activity of the small GTPase Arf6 in cancer cells are potential targets for developing innovative anti-cancer drugs.

PubMed

Yamauchi, Yohei; Miura, Yuki; Kanaho, Yasunori

2017-01-01

The Small GTPase ADP-ribosylation factor 6 (Arf6) functions as the molecular switch in cellular signaling pathways by cycling between GDP-bound inactive and GTP-bound active form, which is precisely regulated by two regulators, guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Numerous studies have shown that these machineries play critical roles in tumor angiogenesis/growth and cancer cell invasion/metastasis through regulating the cycling of Arf6. Here, we summarize accumulating knowledge for involvement of Arf6 GEFs/GAPs and small molecule inhibitors of Arf6 signaling/cycling in cancer progression, and discuss possible strategies for developing innovative anti-cancer drugs targeting Arf6 signaling/cycling. Copyright © 2016 Elsevier Ltd. All rights reserved.

GASS-WEB: a web server for identifying enzyme active sites based on genetic algorithms.

PubMed

Moraes, João P A; Pappa, Gisele L; Pires, Douglas E V; Izidoro, Sandro C

2017-07-03

Enzyme active sites are important and conserved functional regions of proteins whose identification can be an invaluable step toward protein function prediction. Most of the existing methods for this task are based on active site similarity and present limitations including performing only exact matches on template residues, template size restraints, despite not being capable of finding inter-domain active sites. To fill this gap, we proposed GASS-WEB, a user-friendly web server that uses GASS (Genetic Active Site Search), a method based on an evolutionary algorithm to search for similar active sites in proteins. GASS-WEB can be used under two different scenarios: (i) given a protein of interest, to match a set of specific active site templates; or (ii) given an active site template, looking for it in a database of protein structures. The method has shown to be very effective on a range of experiments and was able to correctly identify >90% of the catalogued active sites from the Catalytic Site Atlas. It also managed to achieve a Matthew correlation coefficient of 0.63 using the Critical Assessment of protein Structure Prediction (CASP 10) dataset. In our analysis, GASS was ranking fourth among 18 methods. GASS-WEB is freely available at http://gass.unifei.edu.br/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Sestrin2 is a leucine sensor for the mTORC1 pathway

PubMed Central

Wolfson, Rachel L.; Chantranupong, Lynne; Saxton, Robert A.; Shen, Kuang; Scaria, Sonia M.; Cantor, Jason R.; Sabatini, David M.

2015-01-01

Leucine is a proteogenic amino acid that also regulates many aspects of mammalian physiology, in large part by activating the mTOR complex 1 (mTORC1) protein kinase, a master growth controller. Amino acids signal to mTORC1 through the Rag guanine triphosphatases (GTPases). Several factors regulate the Rags, including GATOR1, a GTPase activating protein (GAP); GATOR2, a positive regulator of unknown function; and Sestrin2, a GATOR2-interacting protein that inhibits mTORC1 signaling. We find that leucine, but not arginine, disrupts the Sestrin2-GATOR2 interaction by binding to Sestrin2 with a Kd of 20 µM, which is the leucine concentration that half-maximally activates mTORC1. The leucine-binding capacity of Sestrin2 is required for leucine to activate mTORC1 in cells. These results indicate that Sestrin2 is a leucine sensor for the mTORC1 pathway. PMID:26449471

Grb2 mediates semaphorin-4D-dependent RhoA inactivation.

PubMed

Sun, Tianliang; Krishnan, Rameshkumar; Swiercz, Jakub M

2012-08-01

Signaling through the semaphorin 4D (Sema4D) receptor plexin-B1 is modulated by its interaction with tyrosine kinases ErbB-2 and Met. In cells expressing the plexin-B1-ErbB-2 receptor complex, ligand stimulation results in the activation of small GTPase RhoA and stimulation of cellular migration. By contrast, in cells expressing plexin-B1 and Met, ligand stimulation results in an association with the RhoGTPase-activating protein p190 RhoGAP and subsequent RhoA inactivation--a process that involves the tyrosine phosphorylation of plexin-B1 by Met. Inactivation of RhoA is necessary for Sema4D-mediated inhibition of cellular migration. It is, however, unknown how plexin-B1 phosphorylation regulates RhoGAP interaction and activity. Here we show that the activation of plexin-B1 by Sema4D and its subsequent tyrosine phosphorylation by Met creates a docking site for the SH2 domain of growth factor receptor bound-2 (Grb2). Grb2 is thereby recruited into the plexin-B1 receptor complex and, through its SH3 domain, interacts with p190 RhoGAP and mediates RhoA deactivation. Phosphorylation of plexin-B1 by Met and the recruitment of Grb2 have no effect on the R-RasGAP activity of plexin-B1, but are required for Sema4D-induced, RhoA-dependent antimigratory effects of Sema4D on breast cancer cells. These data show Grb2 as a direct link between plexin and p190-RhoGAP-mediated downstream signaling.

hnRNP-Q1 represses nascent axon growth in cortical neurons by inhibiting Gap-43 mRNA translation

PubMed Central

Williams, Kathryn R.; McAninch, Damian S.; Stefanovic, Snezana; Xing, Lei; Allen, Megan; Li, Wenqi; Feng, Yue; Mihailescu, Mihaela Rita; Bassell, Gary J.

2016-01-01

Posttranscriptional regulation of gene expression by mRNA-binding proteins is critical for neuronal development and function. hnRNP-Q1 is an mRNA-binding protein that regulates mRNA processing events, including translational repression. hnRNP-Q1 is highly expressed in brain tissue, suggesting a function in regulating genes critical for neuronal development. In this study, we have identified Growth-associated protein 43 (Gap-43) mRNA as a novel target of hnRNP-Q1 and have demonstrated that hnRNP-Q1 represses Gap-43 mRNA translation and consequently GAP-43 function. GAP-43 is a neuronal protein that regulates actin dynamics in growth cones and facilitates axonal growth. Previous studies have identified factors that regulate Gap-43 mRNA stability and localization, but it remains unclear whether Gap-43 mRNA translation is also regulated. Our results reveal that hnRNP-Q1 knockdown increased nascent axon length, total neurite length, and neurite number in mouse embryonic cortical neurons and enhanced Neuro2a cell process extension; these phenotypes were rescued by GAP-43 knockdown. Additionally, we have identified a G-quadruplex structure in the 5′ untranslated region of Gap-43 mRNA that directly interacts with hnRNP-Q1 as a means to inhibit Gap-43 mRNA translation. Therefore hnRNP-Q1–mediated repression of Gap-43 mRNA translation provides an additional mechanism for regulating GAP-43 expression and function and may be critical for neuronal development. PMID:26658614

Restoring functional neurofibromin by protein transduction.

PubMed

Mellert, K; Lechner, S; Lüdeke, M; Lamla, M; Möller, P; Kemkemer, R; Scheffzek, K; Kaufmann, D

2018-04-18

In Neurofibromatosis 1 (NF1) germ line loss of function mutations result in reduction of cellular neurofibromin content (NF1+/-, NF1 haploinsufficiency). The Ras-GAP neurofibromin is a very large cytoplasmic protein (2818 AA, 319 kDa) involved in the RAS-MAPK pathway. Aside from regulation of proliferation, it is involved in mechanosensoric of cells. We investigated neurofibromin replacement in cultured human fibroblasts showing reduced amount of neurofibromin. Full length neurofibromin was produced recombinantly in insect cells and purified. Protein transduction into cultured fibroblasts was performed employing cell penetrating peptides along with photochemical internalization. This combination of transduction strategies ensures the intracellular uptake and the translocation to the cytoplasm of neurofibromin. The transduced neurofibromin is functional, indicated by functional rescue of reduced mechanosensoric blindness and reduced RasGAP activity in cultured fibroblasts of NF1 patients or normal fibroblasts treated by NF1 siRNA. Our study shows that recombinant neurofibromin is able to revert cellular effects of NF1 haploinsuffiency in vitro, indicating a use of protein transduction into cells as a potential treatment strategy for the monogenic disease NF1.

Go in for the kill: How plants deploy effector-triggered immunity to combat pathogens. [Corrected].

PubMed

Wu, Liang; Chen, Huan; Curtis, Chad; Fu, Zheng Qing

2014-01-01

Plant resistance (R) proteins perceive specific pathogen effectors from diverse plant pathogens to initiate defense responses, designated effector-triggered immunity (ETI). Plant R proteins are mostly nucleotide binding-leucine rich repeat (NB-LRR) proteins, which recognize pathogen effectors directly or indirectly through sophisticated mechanisms. Upon activation by effector proteins, R proteins elicit robust defense responses, including a rapid burst of reactive oxygen species (ROS), induced biosynthesis and accumulation of salicylic acid (SA), a rapid programmed cell death (PCD) called hypersensitive response (HR) at the infection sites, and increased expression of pathogenesis-related (PR) genes. Initiation of ETI is correlated with a complex network of defense signaling pathways, resulting in defensive cellular responses and large-scale transcriptional reprogramming events. In this review, we highlight important recent advances on the recognition of effectors, regulation and activation of plant R proteins, dynamic intracellular trafficking of R proteins, induction of cell death, and transcriptional reprogramming associated with ETI. Current knowledge gaps and future research directions are also discussed in this review.

Go in for the kill

PubMed Central

Wu, Liang; Chen, Huan; Curtis, Chad; Fu, Zheng Qing

2014-01-01

Plant resistance (R) proteins perceive specific pathogen effectors from diverse plant pathogens to initiate defense responses, designated effector-triggered immunity (ETI). Plant R proteins are mostly nucleotide binding-leucine rich repeat (NB-LRR) proteins, which recognize pathogen effectors directly or indirectly through sophisticated mechanisms. Upon activation by effector proteins, R proteins elicit robust defense responses, including a rapid burst of reactive oxygen species (ROS), induced biosynthesis and accumulation of salicylic acid (SA), a rapid programmed cell death (PCD) called hypersensitive response (HR) at the infection sites, and increased expression of pathogenesis-related (PR) genes. Initiation of ETI is correlated with a complex network of defense signaling pathways, resulting in defensive cellular responses and large-scale transcriptional reprogramming events. In this review, we highlight important recent advances on the recognition of effectors, regulation and activation of plant R proteins, dynamic intracellular trafficking of R proteins, induction of cell death, and transcriptional reprogramming associated with ETI. Current knowledge gaps and future research directions are also discussed in this review. PMID:25513772

Gap junction protein expression and cellularity: comparison of immature and adult equine digital tendons

PubMed Central

Stanley, Rachael L; Fleck, Roland A; Becker, David L; Goodship, Allen E; Ralphs, Jim R; Patterson-Kane, Janet C

2007-01-01

Injury to the energy-storing superficial digital flexor tendon is common in equine athletes and is age-related. Tenocytes in the superficial digital flexor tendon of adult horses appear to have limited ability to respond adaptively to exercise or prevent the accumulation of strain-induced microdamage. It has been suggested that conditioning exercise should be introduced during the growth period, when tenocytes may be more responsive to increased quantities or intensities of mechanical strain. Tenocytes are linked into networks by gap junctions that allow coordination of synthetic activity and facilitate strain-induced collagen synthesis. We hypothesised that there are reductions in cellular expression of the gap junction proteins connexin (Cx) 43 and 32 during maturation and ageing of the superficial digital flexor tendon that do not occur in the non-injury-prone common digital extensor tendon. Cryosections from the superficial digital flexor tendon and common digital extensor tendon of 5 fetuses, 5 foals (1–6 months), 5 young adults (2–7 years) and 5 old horses (18–33 years) were immunofluorescently labelled and quantitative confocal laser microscopy was performed. Expression of Cx43 and Cx32 protein per tenocyte was significantly higher in the fetal group compared with all other age groups in both tendons. The density of tenocytes was found to be highest in immature tissue. Higher levels of cellularity and connexin protein expression in immature tendons are likely to relate to requirements for tissue remodelling and growth. However, if further studies demonstrate that this correlates with greater gap junctional communication efficiency and synthetic responsiveness to mechanical strain in immature compared with adult tendons, it could support the concept of early introduction of controlled exercise as a means of increasing resistance to later injury. PMID:17848160

Degradation of connexins and gap junctions

PubMed Central

Falk, Matthias M.; Kells, Rachael M.; Berthoud, Viviana M.

2014-01-01

Connexin proteins are short-lived within the cell, whether present in the secretory pathway or in gap junction plaques. Their levels can be modulated by their rate of degradation. Connexins, at different stages of assembly, are degraded through the proteasomal, endo-/lysosomal, and phago-/lysosomal pathways. In this review, we summarize the current knowledge about connexin and gap junction degradation including the signals and protein-protein interactions that participate in their targeting for degradation. PMID:24486527

Death of Neurons following Injury Requires Conductive Neuronal Gap Junction Channels but Not a Specific Connexin

PubMed Central

Fontes, Joseph D.; Ramsey, Jon; Polk, Jeremy M; Koop, Andre; Denisova, Janna V.; Belousov, Andrei B.

2015-01-01

Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury). In cultures prepared from wild-type mice, over-expression and knockdown of Cx36-containing gap junctions augmented and prevented, respectively, neuronal death from NMDAR-mediated excitotoxicity and ischemia. In cultures obtained form from Cx36 knockout mice, re-expression of functional gap junction channels, containing either neuronal Cx36 or non-neuronal Cx43 or Cx31, resulted in increased neuronal death following insult. In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect. Finally, the absence of ethidium bromide uptake in non-transduced wild-type neurons two hours following NMDAR excitotoxicity or ischemia suggested the absence of active endogenous hemichannels in those neurons. Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons. A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed. PMID:26017008

Growth Associated Protein 43 (GAP-43) as a Novel Target for the Diagnosis, Treatment and Prevention of Epileptogenesis.

PubMed

Nemes, Ashley D; Ayasoufi, Katayoun; Ying, Zhong; Zhou, Qi-Gang; Suh, Hoonkyo; Najm, Imad M

2017-12-18

We previously showed increased growth associated protein 43 (GAP-43) expression in brain samples resected from patients with cortical dysplasia (CD), which was correlated with duration of epilepsy. Here, we used a rat model of CD to examine the regulation of GAP-43 in the brain and serum over the course of epileptogenesis. Baseline GAP-43 expression was higher in CD animals compared to control non-CD rats. An acute seizure increased GAP-43 expression in both CD and control rats. However, GAP-43 expression decreased by day 15 post-seizure in control rats, which did not develop spontaneous seizures. In contrast, GAP-43 remained up-regulated in CD rats, and over 50% developed chronic epilepsy with increased GAP-43 levels in their serum. GAP-43 protein was primarily located in excitatory neurons, suggesting its functional significance in epileptogenesis. Inhibition of GAP-43 expression by shRNA significantly reduced seizure duration and severity in CD rats after acute seizures with subsequent reduction in interictal spiking. Serum GAP-43 levels were significantly higher in CD rats that developed spontaneous seizures. Together, these results suggest GAP-43 as a key factor promoting epileptogenesis, a possible therapeutic target for treatment of progressive epilepsy and a potential biomarker for epilepsy progression in CD.

A RabGAP Regulates Life-Cycle Duration via Trimeric G-protein Cascades in Dictyostelium discoideum

PubMed Central

Kuwayama, Hidekazu; Miyanaga, Yukihiro; Urushihara, Hideko; Ueda, Masahiro

2013-01-01

Background The life-cycle of cellular slime molds comprises chronobiologically regulated processes. During the growth phase, the amoeboid cells proliferate at a definite rate. Upon starvation, they synthesize cAMP as both first and second messengers in signalling pathways and form aggregates, migrating slugs, and fruiting bodies, consisting of spores and stalk cells, within 24 h. In Dictyostelium discoideum, because most growth-specific events cease during development, proliferative and heterochronic mutations are not considered to be interrelated and no genetic factor governing the entire life-cycle duration has ever been identified. Methodology/Principal Findings Using yeast 2-hybrid library screening, we isolated a Dictyostelium discoideum RabGAP, Dd Rbg-3, as a candidate molecule by which the Dictyostelium Gα2 subunit directs its effects. Rab GTPase-activating protein, RabGAP, acts as a negative regulator of Rab small GTPases, which orchestrate the intracellular membrane trafficking involved in cell proliferation. Deletion mutants of Dd rbg-3 exhibited an increased growth rate and a shortened developmental period, while an overexpression mutant demonstrated the opposite effects. We also show that Dd Rbg-3 interacts with 2 Gα subunits in an activity-dependent manner in vitro. Furthermore, both human and Caenorhabditis elegans rbg-3 homologs complemented the Dd rbg-3–deletion phenotype in D. discoideum, indicating that similar pathways may be generally conserved in multicellular organisms. Conclusions/Significance Our findings suggest that Dd Rbg-3 acts as a key element regulating the duration of D. discoideum life-span potentially via trimeric G-protein cascades. PMID:24349132

A single high dose of dexamethasone increases GAP-43 and synaptophysin in the hippocampus of aged rats.

PubMed

Tesic, Vesna; Perovic, Milka; Zaletel, Ivan; Jovanovic, Mirna; Puskas, Nela; Ruzdijic, Sabera; Kanazir, Selma

2017-11-01

The administration of dexamethasone, a synthetic glucocorticoid receptor agonist, has been reported to modulate cognitive performance in both animals and humans. In the present study, we demonstrate the effects of a single high dose of dexamethasone on the expression and distribution of synaptic plasticity-related proteins, growth-associated protein-43 (GAP-43) and synaptophysin, in the hippocampus of 6-, 12-, 18- and 24-month-old rats. Acute dexamethasone treatment significantly altered the expression of GAP-43 at the posttranslational level by modulating the levels of phosphorylated GAP-43 and proteolytic GAP-43-3 fragment. The effect was the most pronounced in the hippocampi of the aged animals. The total GAP-43 protein was increased only in 24-month-old dexamethasone-treated animals, and was concomitant with a decrease in calpain-mediated proteolysis. Moreover, by introducing the gray level co-occurrence matrix method, a form of texture analysis, we were able to reveal the subtle differences in the expression pattern of both GAP-43 and synaptophysin in the hippocampal subfields that were not detected by Western blot analysis alone. Therefore, the current study demonstrates, through a novel combined approach, that dexamethasone treatment significantly affects both GAP-43 and synaptophysin protein expression in the hippocampus of aged rats. Copyright © 2017 Elsevier Inc. All rights reserved.

Variations in gap junctional intercellular communication and connexin expression in fibroblasts derived from keloid and hypertrophic scars.

PubMed

Lu, Feng; Gao, JianHua; Ogawa, Rei; Hyakusoku, Hiko

2007-03-01

Expression of connexins and other constituent proteins of gap junctions along with gap junctional intercellular communication are involved in cellular development and differentiation processes. In addition, an increasing number of hereditary skin disorders appear to be linked to connexins. Therefore, in this report, the authors studied in vitro gap junctional intercellular communication function and connexin expression in fibroblasts derived from keloid and hypertrophic scar patients. Fibroblasts harvested from each of six keloid and hypertrophic scar patients were used for this study. Gap junctional intercellular communication function was investigated using the gap fluorescence recovery after photobleaching method, and expression of connexin proteins was studied using quantitative confocal microscopic analyses. Compared with normal skin, a decreased level of gap junctional intercellular communication was seen in fibroblasts derived from hypertrophic scar tissue, whereas an extremely low gap junctional intercellular communication level was detected in fibroblasts derived from keloid tissue. We also detected little connexin 43 (Cx43) protein localized in fibroblasts derived from keloids. Moreover, Cx43 protein levels were much lower in fibroblasts derived from hypertrophic scars than in those derived from normal skin. The authors' data suggest that the loss of gap junctional intercellular communication and connexin expression may affect intercellular recognition and thus break the proliferation and apoptosis balance in fibroblasts derived from keloid and hypertrophic scar tissue.

The influence of GAP-43 on orientation of cell division through G proteins.

PubMed

Huang, Rui; Zhao, Junpeng; Ju, Lili; Wen, Yujun; Xu, Qunyuan

2015-12-01

Recent studies have shown that GAP-43 is highly expressed in horizontally dividing neural progenitor cells, and G protein complex are required for proper mitotic-spindle orientation of those progenitors in the mammalian developing cortex. In order to verify the hypothesis that GAP-43 may influence the orientation of cell division through interacting with G proteins during neurogenesis, the GAP-43 RNA from adult C57 mouse was cloned into the pEGFP-N1 vector, which was then transfected into Madin-Darby Canine Kidney (MDCK) cells cultured in a three-dimensional (3D) cell culture system. The interaction of GAP-43 with Gαi was detected by co-immunoprecipitation (co-IP), while cystogenesis of 3D morphogenesis of MDCK cells and expression of GAP-43 and Gαi were determined by immunofluorescence and Western blotting. The results showed are as follows: After being transfected by pEGFP-N1-GAP-43, GAP-43 was localized on the cell membrane and co-localized with Gαi, and this dramatically induced a defective cystogenesis in 3D morphogenesis of MDCK cells. The functional interaction between GAP-43 and Gαi proteins was proven by the co-IP assay. It can be considered from the results that the GAP-43 is involved in the orientation of cell division by interacting with Gαi and this should be an important mechanism for neurogenesis in the mammalian brain. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cell division requires a direct link between microtubule-bound RacGAP and Anillin in the contractile ring.

PubMed

Gregory, Stephen L; Ebrahimi, Saman; Milverton, Joanne; Jones, Whitney M; Bejsovec, Amy; Saint, Robert

2008-01-08

The mitotic microtubule array plays two primary roles in cell division. It acts as a scaffold for the congression and separation of chromosomes, and it specifies and maintains the contractile-ring position. The current model for initiation of Drosophila and mammalian cytokinesis [1-5] postulates that equatorial localization of a RhoGEF (Pbl/Ect2) by a microtubule-associated motor protein complex creates a band of activated RhoA [6], which subsequently recruits contractile-ring components such as actin, myosin, and Anillin [1-3]. Equatorial microtubules are essential for continued constriction, but how they interact with the contractile apparatus is unknown. Here, we report the first direct molecular link between the microtubule spindle and the actomyosin contractile ring. We find that the spindle-associated component, RacGAP50C, which specifies the site of cleavage [1-5], interacts directly with Anillin, an actin and myosin binding protein found in the contractile ring [7-10]. Both proteins depend on this interaction for their localization. In the absence of Anillin, the spindle-associated RacGAP loses its association with the equatorial cortex, and cytokinesis fails. These results account for the long-observed dependence of cytokinesis on the continual presence of microtubules at the cortex.

Network of Surface-Displayed Glycolytic Enzymes in Mycoplasma pneumoniae and Their Interactions with Human Plasminogen

PubMed Central

Gründel, Anne; Pfeiffer, Melanie; Jacobs, Enno

2015-01-01

In different bacteria, primarily cytosolic and metabolic proteins are characterized as surface localized and interacting with different host factors. These moonlighting proteins include glycolytic enzymes, and it has been hypothesized that they influence the virulence of pathogenic species. The presence of surface-displayed glycolytic enzymes and their interaction with human plasminogen as an important host factor were investigated in the genome-reduced and cell wall-less microorganism Mycoplasma pneumoniae, a common agent of respiratory tract infections of humans. After successful expression of 19 glycolytic enzymes and production of polyclonal antisera, the localization of proteins in the mycoplasma cell was characterized using fractionation of total proteins, colony blot, mild proteolysis and immunofluorescence of M. pneumoniae cells. Eight glycolytic enzymes, pyruvate dehydrogenases A to C (PdhA-C), glyceraldehyde-3-phosphate dehydrogenase (GapA), lactate dehydrogenase (Ldh), phosphoglycerate mutase (Pgm), pyruvate kinase (Pyk), and transketolase (Tkt), were confirmed as surface expressed and all are able to interact with plasminogen. Plasminogen bound to recombinant proteins PdhB, GapA, and Pyk was converted to plasmin in the presence of urokinase plasminogen activator and plasmin-specific substrate d-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. Furthermore, human fibrinogen was degraded by the complex of plasminogen and recombinant protein PdhB or Pgm. In addition, surface-displayed proteins (except PdhC) bind to human lung epithelial cells, and the interaction was reduced significantly by preincubation of cells with antiplasminogen. Our results suggest that plasminogen binding and activation by different surface-localized glycolytic enzymes of M. pneumoniae may play a role in successful and long-term colonization of the human respiratory tract. PMID:26667841

GAP-43 Gene Expression Regulates Information Storage

ERIC Educational Resources Information Center

Holahan, Matthew R.; Honegger, Kyle S.; Tabatadze, Nino; Routtenberg, Aryeh

2007-01-01

Previous reports have shown that overexpression of the growth- and plasticity-associated protein GAP-43 improves memory. However, the relation between the levels of this protein to memory enhancement remains unknown. Here, we studied this issue in transgenic mice (G-Phos) overexpressing native, chick GAP-43. These G-Phos mice could be divided at…

The RhoGAP SPIN6 Associates with SPL11 and OsRac1 and Negatively Regulates Programmed Cell Death and Innate Immunity in Rice

PubMed Central

Liu, Jinling; Park, Chan Ho; He, Feng; Nagano, Minoru; Wang, Mo; Bellizzi, Maria; Zhang, Kai; Zeng, Xiaoshan; Liu, Wende; Ning, Yuese; Kawano, Yoji; Wang, Guo-Liang

2015-01-01

The ubiquitin proteasome system in plants plays important roles in plant-microbe interactions and in immune responses to pathogens. We previously demonstrated that the rice U-box E3 ligase SPL11 and its Arabidopsis ortholog PUB13 negatively regulate programmed cell death (PCD) and defense response. However, the components involved in the SPL11/PUB13-mediated PCD and immune signaling pathway remain unknown. In this study, we report that SPL11-interacting Protein 6 (SPIN6) is a Rho GTPase-activating protein (RhoGAP) that interacts with SPL11 in vitro and in vivo. SPL11 ubiquitinates SPIN6 in vitro and degrades SPIN6 in vivo via the 26S proteasome-dependent pathway. Both RNAi silencing in transgenic rice and knockout of Spin6 in a T-DNA insertion mutant lead to PCD and increased resistance to the rice blast pathogen Magnaporthe oryzae and the bacterial blight pathogen Xanthomonas oryzae pv. oryzae. The levels of reactive oxygen species and defense-related gene expression are significantly elevated in both the Spin6 RNAi and mutant plants. Strikingly, SPIN6 interacts with the small GTPase OsRac1, catalyze the GTP-bound OsRac1 into the GDP-bound state in vitro and has GAP activity towards OsRac1 in rice cells. Together, our results demonstrate that the RhoGAP SPIN6 acts as a linkage between a U-box E3 ligase-mediated ubiquitination pathway and a small GTPase-associated defensome system for plant immunity. PMID:25658451

Mitochondrial protein import: a matter of death?

PubMed

Paschen, Stefan A; Weber, Arnim; Häcker, Georg

2007-10-15

Mitochondria play a central role not only in energy generation but also for apoptosis. A key step in mitochondrial apoptosis is the release of mitochondrial proteins, most importantly cytochrome c. This release is orchestrated by the pro- and anti-apoptotic members of the Bcl-2 protein family. The functions of these Bcl-2 family members are clear in terms of order and of principle: the pro-apoptotic BH3-only protein group contains the triggers, which cause the activation of the effectors Bax and Bak, while the anti-apoptotic Bcl-2-like proteins prevent this activation. However, the molecular details are still insufficiently clear and the proposed models have certain gaps and are partly contradictory. We have recently presented evidence that targeting to mitochondria of at least one BH3-only protein is essential for its pro-apoptotic functions. Here we discuss how this mechanism might fit into and expand existing models and speculate about the potential implications of this finding.

Cellular Interaction of Integrin α3β1 with Laminin 5 Promotes Gap Junctional Communication

PubMed Central

Lampe, Paul D.; Nguyen, Beth P.; Gil, Susana; Usui, Marcia; Olerud, John; Takada, Yoshikazu; Carter, William G.

1998-01-01

Wounding of skin activates epidermal cell migration over exposed dermal collagen and fibronectin and over laminin 5 secreted into the provisional basement membrane. Gap junctional intercellular communication (GJIC) has been proposed to integrate the individual motile cells into a synchronized colony. We found that outgrowths of human keratinocytes in wounds or epibole cultures display parallel changes in the expression of laminin 5, integrin α3β1, E-cadherin, and the gap junctional protein connexin 43. Adhesion of keratinocytes on laminin 5, collagen, and fibronectin was found to differentially regulate GJIC. When keratinocytes were adhered on laminin 5, both structural (assembly of connexin 43 in gap junctions) and functional (dye transfer) assays showed a two- to threefold increase compared with collagen and five- to eightfold over fibronectin. Based on studies with immobilized integrin antibody and integrin-transfected Chinese hamster ovary cells, the interaction of integrin α3β1 with laminin 5 was sufficient to promote GJIC. Mapping of intermediate steps in the pathway linking α3β1–laminin 5 interactions to GJIC indicated that protein trafficking and Rho signaling were both required. We suggest that adhesion of epithelial cells to laminin 5 in the basement membrane via α3β1 promotes GJIC that integrates individual cells into synchronized epiboles. PMID:9852164

Ankyrin-B is a PI3P effector that promotes polarized α5β1-integrin recycling via recruiting RabGAP1L to early endosomes

PubMed Central

Qu, Fangfei; Lorenzo, Damaris N; King, Samantha J; Brooks, Rebecca; Bear, James E; Bennett, Vann

2016-01-01

Endosomal membrane trafficking requires coordination between phosphoinositide lipids, Rab GTPases, and microtubule-based motors to dynamically determine endosome identity and promote long-range organelle transport. Here we report that ankyrin-B (AnkB), through integrating all three systems, functions as a critical node in the protein circuitry underlying polarized recycling of α5β1-integrin in mouse embryonic fibroblasts, which enables persistent fibroblast migration along fibronectin gradients. AnkB associates with phosphatidylinositol 3-phosphate (PI3P)-positive organelles in fibroblasts and binds dynactin to promote their long-range motility. We demonstrate that AnkB binds to Rab GTPase Activating Protein 1-Like (RabGAP1L) and recruits it to PI3P-positive organelles, where RabGAP1L inactivates Rab22A, and promotes polarized trafficking to the leading edge of migrating fibroblasts. We further determine that α5β1-integrin depends on an AnkB/RabGAP1L complex for polarized recycling. Our results reveal AnkB as an unexpected key element in coordinating polarized transport of α5β1-integrin and likely of other specialized endocytic cargos. DOI: http://dx.doi.org/10.7554/eLife.20417.001 PMID:27718357

Hydrogen Sulfide Regulates the Cytosolic/Nuclear Partitioning of Glyceraldehyde-3-Phosphate Dehydrogenase by Enhancing its Nuclear Localization.

PubMed

Aroca, Angeles; Schneider, Markus; Scheibe, Renate; Gotor, Cecilia; Romero, Luis C

2017-06-01

Hydrogen sulfide is an important signaling molecule comparable with nitric oxide and hydrogen peroxide in plants. The underlying mechanism of its action is unknown, although it has been proposed to be S-sulfhydration. This post-translational modification converts the thiol groups of cysteines within proteins to persulfides, resulting in functional changes of the proteins. In Arabidopsis thaliana, S-sulfhydrated proteins have been identified, including the cytosolic isoforms of glyceraldehyde-3-phosphate dehydrogenase GapC1 and GapC2. In this work, we studied the regulation of sulfide on the subcellular localization of these proteins using two different approaches. We generated GapC1-green fluorescent protein (GFP) and GapC2-GFP transgenic plants in both the wild type and the des1 mutant defective in the l-cysteine desulfhydrase DES1, responsible for the generation of sulfide in the cytosol. The GFP signal was detected in the cytoplasm and the nucleus of epidermal cells, although with reduced nuclear localization in des1 compared with the wild type, and exogenous sulfide treatment resulted in similar signals in nuclei in both backgrounds. The second approach consisted of the immunoblot analysis of the GapC endogenous proteins in enriched nuclear and cytosolic protein extracts, and similar results were obtained. A significant reduction in the total amount of GapC in des1 in comparison with the wild type was determined and exogenous sulfide significantly increased the protein levels in the nuclei in both plants, with a stronger response in the wild type. Moreover, the presence of an S-sulfhydrated cysteine residue on GapC1 was demonstrated by mass spectrometry. We conclude that sulfide enhances the nuclear localization of glyceraldehyde-3-phosphate dehydrogenase. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

RacGAP50C is sufficient to signal cleavage furrow formation during cytokinesis.

PubMed

D'Avino, Pier Paolo; Savoian, Matthew S; Capalbo, Luisa; Glover, David M

2006-11-01

Several studies indicate that spindle microtubules determine the position of the cleavage plane at the end of cell division, but their exact role in triggering the formation and ingression of the cleavage furrow is still unclear. Here we show that in Drosophila depletion of either the GAP (GTPase-activating protein) or the kinesin-like subunit of the evolutionary conserved centralspindlin complex prevents furrowing without affecting the association of astral microtubules with the cell cortex. Moreover, time-lapse imaging indicates that astral microtubules serve to deliver the centralspindlin complex to the equatorial cortex just before furrow formation. However, when the GAP-signaling component was mislocalized around the entire cortex using a membrane-tethering motif, this caused ectopic furrowing even in the absence of its motor partner. Thus, the GAP component of centralspindlin is both necessary and sufficient for furrow formation and ingression and astral microtubules provide a route for its delivery to the cleavage site.

Role of pp60(c-src) and p(44/42) MAPK in ANG II-induced contraction of rat tonic gastrointestinal smooth muscles.

PubMed

Puri, Rajinder N; Fan, Ya-Ping; Rattan, Satish

2002-08-01

We examined the role of mitogen-activated protein kinase (p(44/42) MAPK) in ANG II-induced contraction of lower esophageal sphincter (LES) and internal anal sphincter (IAS) smooth muscles. Studies were performed in the isolated smooth muscles and cells (SMC). ANG II-induced changes in the levels of phosphorylation of different signal transduction and effector proteins were determined before and after selective inhibitors. ANG II-induced contraction of the rat LES and IAS SMC was inhibited by genistein, PD-98059 [a specific inhibitor of MAPK kinases (MEK 1/2)], herbimycin A (a pp60(c-src) inhibitor), and antibodies to pp60(c-src) and p(120) ras GTPase-activating protein (p(120) rasGAP). ANG II-induced contraction of the tonic smooth muscles was accompanied by an increase in tyrosine phosphorylation of p(120) rasGAP. These were attenuated by genistein but not by PD-98059. ANG II-induced increase in phosphorylations of p(44/42) MAPKs and caldesmon was attenuated by both genistein and PD-98059. We conclude that pp60(c-src) and p(44/42) MAPKs play an important role in ANG II-induced contraction of LES and IAS smooth muscles.

Rho proteins of plants--functional cycle and regulation of cytoskeletal dynamics.

PubMed

Mucha, Elena; Fricke, Inka; Schaefer, Antje; Wittinghofer, Alfred; Berken, Antje

2011-11-01

Rho-related ROP proteins are molecular switches that essentially regulate a wide variety of processes. Of central interest is their influence on the plant cytoskeleton by which they affect vital processes like cell division, growth, morphogenesis, and pathogen defense. ROPs switch between GTP- and GDP-bound conformations by strictly regulated nucleotide exchange and GTP-hydrolysis, and only the active GTP-form interacts with downstream effectors to ultimately provoke a biological response. However, the mode of action of the engaged regulators and effectors as well as their upstream and downstream interaction partners have long been largely unknown. As opposed to analogous systems in animals and fungi, plants use specific GTPase activating proteins (RopGAPs) with a unique domain composition and novel guanine nucleotide exchange factors (RopGEFs) with a probable link to cell surface receptors. Moreover, plants comprise novel effector molecules and adapters connecting ROPs to mostly unknown downstream targets on the route to the cytoskeleton. This review aims to summarize recent knowledge on the molecular mechanisms and reaction cascades involved in ROP dependent cytoskeletal rearrangements, addressing the structure and function of the unusual RopGAPs, RopGEFs and effectors, and the upstream and downstream pathways linking ROPs to cell receptor-like kinases, actin filaments, and microtubules. Copyright © 2010 Elsevier GmbH. All rights reserved.

Opportunities and Challenges for Nutritional Proteomics in Cancer Prevention12

PubMed Central

Romagnolo, Donato F.; Milner, John A.

2012-01-01

Knowledge gaps persist about the efficacy of cancer prevention strategies based on dietary food components. Adaptations to nutrient supply are executed through tuning of multiple protein networks that include transcription factors, histones, modifying enzymes, translation factors, membrane and nuclear receptors, and secreted proteins. However, the simultaneous quantitative and qualitative measurement of all proteins that regulate cancer processes is not practical using traditional protein methodologies. Proteomics offers an attractive opportunity to fill this knowledge gap and unravel the effects of dietary components on protein networks that impinge on cancer. The articles presented in this supplement are from talks proffered in the “Nutrition Proteomics and Cancer Prevention” session at the American Institute for Cancer Research Annual Research Conference on Food, Nutrition, Physical Activity and Cancer held in Washington, DC on October 21 and 22, 2010. Recent advances in MS technologies suggest that studies in nutrition and cancer prevention may benefit from the adoption of proteomic tools to elucidate the impact on biological processes that govern the transition from normal to malignant phenotype; to identify protein changes that determine both positive and negative responses to food components; to assess how protein networks mediate dose-, time-, and tissue-dependent responses to food components; and, finally, for predicting responders and nonresponders. However, both the limited accessibility to proteomic technologies and research funding appear to be hampering the routine adoption of proteomic tools in nutrition and cancer prevention research. PMID:22649262

tif-1 mutation alters polynucleotide recognition by the recA protein of Escherichia coli.

PubMed Central

McEntee, K; Weinstock, G M

1981-01-01

The requirements for polynucleotide-dependent hydrolysis of ATP and for proteolytic cleavage of phage lambda repressor have been examined for both the wild-type (recA+ protein) and the tif-1 mutant form [tif(recA) protein] of the recA gene product. The recA+ and tif(recA) proteins catalyze both reactions in the presence of long single-stranded DNAs or certain deoxyhomopolymers. However, short oligonucleotides [(dT)12, (dA)14] stimulate neither the protease nor the ATPase activities of the recA+ protein. In contrast, these short oligonucleotides activate tif(recA) protein to cleave lambda repressor without stimulating its ATPase activity. Moreover, both the ATPase and protease activities of the tif(recA) protein are stimulated by poly(rU) and poly(rC) whereas the recA+ protein does not respond to these ribopolymers. We have purified the recA protein from a strain in which the tif mutation is intragenically suppressed. This mutant protein (recA629) is inactive in the presence of (dT)12, (dA)14, poly(rU), and poly(rC) for lambda repressor cleavage and ATP hydrolysis. These results argue that the tif-1 mutation (or mutations) alters the DNA binding site of the recA protein. We suggest that in vivo the tif(recA) protein is activated for cleaving repressors of SOS genes by complex formation with short single-stranded regions or gaps that normally occur near the growing fork of replicating chromosomes and are too short for activating the recA+ enzyme. This mechanism can account for the expression of SOS functions in the absence of DNA damage in tif mutant strains. Images PMID:7031642

Endosomal sorting of GLUT4 and Gap1 is conserved between yeast and insulin-sensitive cells

PubMed Central

Shewan, Annette M.; McCann, Rebecca K.; Lamb, Christopher A.; Stirrat, Laura; Kioumourtzoglou, Dimitrios; Adamson, Iain S.; Verma, Suzie; James, David E.; Bryant, Nia J.

2013-01-01

Summary The insulin-regulated trafficking of the facilitative glucose transporter GLUT4 in human fat and muscle cells and the nitrogen-regulated trafficking of the general amino acid permease Gap1 in the yeast Saccharomyces cerevisiae share several common features: Both Gap1 and GLUT4 are nutrient transporters that are mobilised to the cell surface from an intracellular store in response to an environmental cue; both are polytopic membrane proteins harbouring amino acid targeting motifs in their C-terminal tails that are required for their regulated trafficking; ubiquitylation of both Gap1 and GLUT4 plays an important role in their regulated trafficking, as do the ubiquitin-binding GGA (Golgi-localised, γ-ear-containing, ARF-binding) adaptor proteins. Here, we find that when expressed heterologously in yeast, human GLUT4 is subject to nitrogen-regulated trafficking in an ubiquitin-dependent manner similar to Gap1. In addition, by expressing a GLUT4/Gap1 chimeric protein in adipocytes we show that the carboxy-tail of Gap1 directs intracellular sequestration and insulin-regulated trafficking in adipocytes. These findings demonstrate that the trafficking signals and their cognate molecular regulatory machinery that mediate regulated exocytosis of membrane proteins are conserved across evolution. PMID:23424197

Toxicity challenges in environmental chemicals: Prediction of human plasma protein binding through quantitative structure-activity relationship (QSAR) models (2016 IVIVE Workshop Proceedings)

EPA Science Inventory

Physiologically based pharmacokinetic (PBPK) models bridge the gap between in vitro assays and in vivo effects by accounting for the adsorption, distribution, metabolism, and excretion of xenobiotics, which is especially useful in the assessment of human toxicity. Quantitative st...

The CASTOR proteins are arginine sensors for the mTORC1 pathway

PubMed Central

Chantranupong, Lynne; Scaria, Sonia M.; Saxton, Robert A.; Gygi, Melanie P.; Shen, Kuang; Wyant, Gregory A.; Wang, Tim; Harper, J. Wade; Gygi, Steven P.; Sabatini, David M.

2016-01-01

Amino acids signal to the mTOR complex I (mTORC1) growth pathway through the Rag GTPases. Multiple distinct complexes regulate the Rags, including GATOR1, a GTPase activating protein (GAP), and GATOR2, a positive regulator of unknown molecular function. Arginine stimulation of cells activates mTORC1, but how it is sensed is not well understood. Recently, SLC38A9 was identified as a putative lysosomal arginine sensor required for arginine to activate mTORC1 but how arginine deprivation represses mTORC1 is unknown. Here, we show that CASTOR1, a previously uncharacterized protein, interacts with GATOR2 and is required for arginine deprivation to inhibit mTORC1. CASTOR1 homodimerizes and can also heterodimerize with the related protein, CASTOR2. Arginine disrupts the CASTOR1-GATOR2 complex by binding to CASTOR1 with a dissociation constant of ~30 μM, and its arginine-binding capacity is required for arginine to activate mTORC1 in cells. Collectively, these results establish CASTOR1 as an arginine sensor for the mTORC1 pathway. PMID:26972053

[Effects of Chinese herbal compound for supplementing qi and activating blood circulation on actin, Cx43 expressions and gap junctional intercellular communication functions of myocardial cells in patients with Coxsackie virus B 3 viral myocarditis].

PubMed

Zhang, Ming-xue; He, Wei; Gu, Ping

2010-08-01

To observe the effect of Chinese herbal compound for supplementing qi and activating blood circulation (CHC) on the gap junctional intercellular communication (GJIC) function of myocardial cells in patients with Coxsackie virus B 3 (CVB3) viral myocarditis. Expressions of actin and connexin43 (Cx43) in myocardial cells of patients arranged in three groups (the normal control group, the viral infected group and the CHC treated group) were detected by immunohistochemical method; the fluorescence photobleaching recovery rate of cells was detected by laser scanning confocal microscope. As compared with the viral infected group, the expressions of actin and Cx43 were increased and the GJIC function was improved in the CHC treated group. CHC could antagonize viral injury on skeleton protein, and repair the structure of gap junction channel to improve the GJIC function of myocardial cells after being attacked by CVB3.

Proteomics reveals novel components of the Anopheles gambiae eggshell

PubMed Central

Amenya, Dolphine A.; Chou, Wayne; Li, Jianyong; Yan, Guiyun; Gershon, Paul D.; James, Anthony A.; Marinotti, Osvaldo

2010-01-01

While genome and transcriptome sequencing has revealed a large number and diversity of Anopheles gambiae predicted proteins, identifying their functions and biosynthetic pathways remains challenging. Applied mass spectrometry based proteomics in conjunction with mosquito genome and transcriptome databases were used to identify 44 proteins as putative components of the eggshell. Among the identified molecules are two vitelline membrane proteins and a group of seven putative chorion proteins. Enzymes with peroxidase, laccase and phenoloxidase activities, likely involved in cross-linking reactions that stabilize the eggshell structure, also were identified. Seven odorant binding proteins were found in association with the mosquito eggshell, although their role has yet to be demonstrated. This analysis fills a considerable gap of knowledge about proteins that build the eggshell of anopheline mosquitoes. PMID:20433845

Regulation of gap junction conductance by calcineurin through Cx43 phosphorylation: implications for action potential conduction.

PubMed

Jabr, Rita I; Hatch, Fiona S; Salvage, Samantha C; Orlowski, Alejandro; Lampe, Paul D; Fry, Christopher H

2016-11-01

Cardiac arrhythmias are associated with raised intracellular [Ca 2+ ] and slowed action potential conduction caused by reduced gap junction (GJ) electrical conductance (Gj). Ventricular GJs are composed of connexin proteins (Cx43), with Gj determined by Cx43 phosphorylation status. Connexin phosphorylation is an interplay between protein kinases and phosphatases but the precise pathways are unknown. We aimed to identify key Ca 2+ -dependent phosphorylation sites on Cx43 that regulate cardiac gap junction conductance and action potential conduction velocity. We investigated the role of the Ca 2+ -dependent phosphatase, calcineurin. Intracellular [Ca 2+ ] was raised in guinea-pig myocardium by a low-Na solution or increased stimulation. Conduction velocity and Gj were measured in multicellular strips. Phosphorylation of Cx43 serine residues (S365 and S368) and of the intermediary regulator I1 at threonine35 was measured by Western blot. Measurements were made in the presence and absence of inhibitors to calcineurin, I1 or protein phosphatase-1 and phosphatase-2.Raised [Ca 2 + ] i decreased Gj, reduced Cx43 phosphorylation at S365 and increased it at S368; these changes were reversed by calcineurin inhibitors. Cx43-S368 phosphorylation was reversed by the protein kinase C inhibitor chelerythrine. Raised [Ca 2+ ] i also decreased I1 phosphorylation, also prevented by calcineurin inhibitors, to increase activity of the Ca 2+ -independent phosphatase, PPI. The PP1 inhibitor, tautomycin, prevented Cx43-365 dephosphorylation, Cx43-S368 phosphorylation and Gj reduction in raised [Ca 2+ ] i . PP2A had no role. Conduction velocity was reduced by raised [Ca 2+ ] i and reversed by calcineurin inhibitors. Reduced action potential conduction and Gj in raised [Ca 2+ ] are regulated by calcineurin-dependent Cx43-S365 phosphorylation, leading to Cx43-S368 dephosphorylation. The calcineurin action is indirect, via I1 dephosphorylation and subsequent activation of PP1.

Gap Junction Coupling and Calcium Waves in the Pancreatic Islet

PubMed Central

Benninger, Richard K. P.; Zhang, Min; Head, W. Steven; Satin, Leslie S.; Piston, David W.

2008-01-01

The pancreatic islet is a highly coupled, multicellular system that exhibits complex spatiotemporal electrical activity in response to elevated glucose levels. The emergent properties of islets, which differ from those arising in isolated islet cells, are believed to arise in part by gap junctional coupling, but the mechanisms through which this coupling occurs are poorly understood. To uncover these mechanisms, we have used both high-speed imaging and theoretical modeling of the electrical activity in pancreatic islets under a reduction in the gap junction mediated electrical coupling. Utilizing islets from a gap junction protein connexin 36 knockout mouse model together with chemical inhibitors, we can modulate the electrical coupling in the islet in a precise manner and quantify this modulation by electrophysiology measurements. We find that after a reduction in electrical coupling, calcium waves are slowed as well as disrupted, and the number of cells showing synchronous calcium oscillations is reduced. This behavior can be reproduced by computational modeling of a heterogeneous population of β-cells with heterogeneous levels of electrical coupling. The resulting quantitative agreement between the data and analytical models of islet connectivity, using only a single free parameter, reveals the mechanistic underpinnings of the multicellular behavior of the islet. PMID:18805925

The Arf GAP CNT-2 regulates the apoptotic fate in C. elegans asymmetric neuroblast divisions.

PubMed

Singhvi, Aakanksha; Teuliere, Jerome; Talavera, Karla; Cordes, Shaun; Ou, Guangshuo; Vale, Ronald D; Prasad, Brinda C; Clark, Scott G; Garriga, Gian

2011-06-07

During development, all cells make the decision to live or die. Although the molecular mechanisms that execute the apoptotic program are well defined, less is known about how cells decide whether to live or die. In C. elegans, this decision is linked to how cells divide asymmetrically [1, 2]. Several classes of molecules are known to regulate asymmetric cell divisions in metazoans, yet these molecules do not appear to control C. elegans divisions that produce apoptotic cells [3]. We identified CNT-2, an Arf GTPase-activating protein (GAP) of the AGAP family, as a novel regulator of this type of neuroblast division. Loss of CNT-2 alters daughter cell size and causes the apoptotic cell to adopt the fate of its sister cell, resulting in extra neurons. CNT-2's Arf GAP activity is essential for its function in these divisions. The N terminus of CNT-2, which contains a GTPase-like domain that defines the AGAP class of Arf GAPs, negatively regulates CNT-2's function. We provide evidence that CNT-2 regulates receptor-mediated endocytosis and consider the implications of its role in asymmetric cell divisions. Copyright © 2011 Elsevier Ltd. All rights reserved.

Managing the complexity of communication: regulation of gap junctions by post-translational modification

PubMed Central

Axelsen, Lene N.; Calloe, Kirstine; Holstein-Rathlou, Niels-Henrik; Nielsen, Morten S.

2013-01-01

Gap junctions are comprised of connexins that form cell-to-cell channels which couple neighboring cells to accommodate the exchange of information. The need for communication does, however, change over time and therefore must be tightly controlled. Although the regulation of connexin protein expression by transcription and translation is of great importance, the trafficking, channel activity and degradation are also under tight control. The function of connexins can be regulated by several post translational modifications, which affect numerous parameters; including number of channels, open probability, single channel conductance or selectivity. The most extensively investigated post translational modifications are phosphorylations, which have been documented in all mammalian connexins. Besides phosphorylations, some connexins are known to be ubiquitinated, SUMOylated, nitrosylated, hydroxylated, acetylated, methylated, and γ-carboxyglutamated. The aim of the present review is to summarize our current knowledge of post translational regulation of the connexin family of proteins. PMID:24155720

Presence of an SH2 domain in the actin-binding protein tensin.

PubMed

Davis, S; Lu, M L; Lo, S H; Lin, S; Butler, J A; Druker, B J; Roberts, T M; An, Q; Chen, L B

1991-05-03

The molecular cloning of the complementary DNA coding for a 90-kilodalton fragment of tensin, an actin-binding component of focal contacts and other submembraneous cytoskeletal structures, is reported. The derived amino acid sequence revealed the presence of a Src homology 2 (SH2) domain. This domain is shared by a number of signal transduction proteins including nonreceptor tyrosine kinases such as Abl, Fps, Src, and Src family members, the transforming protein Crk, phospholipase C-gamma 1, PI-3 (phosphatidylinositol) kinase, and guanosine triphosphatase-activating protein (GAP). Like the SH2 domain found in Src, Crk, and Abl, the SH2 domain of tensin bound specifically to a number of phosphotyrosine-containing proteins from v-src-transformed cells. Tensin was also found to be phosphorylated on tyrosine residues. These findings suggest that by possessing both actin-binding and phosphotyrosine-binding activities and being itself a target for tyrosine kinases, tensin may link signal transduction pathways with the cytoskeleton.

Protein unfolding as a switch from self-recognition to high-affinity client binding

PubMed Central

Groitl, Bastian; Horowitz, Scott; Makepeace, Karl A. T.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.; Reichmann, Dana; Bardwell, James C. A.; Jakob, Ursula

2016-01-01

Stress-specific activation of the chaperone Hsp33 requires the unfolding of a central linker region. This activation mechanism suggests an intriguing functional relationship between the chaperone's own partial unfolding and its ability to bind other partially folded client proteins. However, identifying where Hsp33 binds its clients has remained a major gap in our understanding of Hsp33's working mechanism. By using site-specific Fluorine-19 nuclear magnetic resonance experiments guided by in vivo crosslinking studies, we now reveal that the partial unfolding of Hsp33's linker region facilitates client binding to an amphipathic docking surface on Hsp33. Furthermore, our results provide experimental evidence for the direct involvement of conditionally disordered regions in unfolded protein binding. The observed structural similarities between Hsp33's own metastable linker region and client proteins present a possible model for how Hsp33 uses protein unfolding as a switch from self-recognition to high-affinity client binding. PMID:26787517

Nuclear translocation of PKCα isoenzyme is involved in neurogenic commitment of human neural crest-derived periodontal ligament stem cells.

PubMed

Trubiani, Oriana; Guarnieri, Simone; Diomede, Francesca; Mariggiò, Maria A; Merciaro, Ilaria; Morabito, Caterina; Cavalcanti, Marcos F X B; Cocco, Lucio; Ramazzotti, Giulia

2016-11-01

Stem cells isolated from human adult tissue niche represent a promising source for neural differentiation. Human Periodontal Ligament Stem Cells (hPDLSCs) originating from the neural crest are particularly suitable for induction of neural commitment. In this study, under xeno-free culture conditions, in undifferentiated hPDLSCs and in hPDLSCs induced to neuronal differentiation by basic Fibroblast Growth Factor, the level of some neural markers have been analyzed. The hPDLSCs spontaneously express Nestin, a neural progenitor marker. In these cells, the neurogenic process induced to rearrange the cytoskeleton, form neurospheres and express higher levels of Nestin and Tyrosine Hydroxylase, indicating neural induction. Protein Kinase C (PKC) is highly expressed in neural tissue and has a key role in neuronal functions. In particular the Ca(2+) and diacylglycerol-dependent activation of PKCα isozyme is involved in the regulation of neuronal differentiation. Another main component of the pathways controlling neuronal differentiation is the Growth Associated Protein-43 (GAP-43), whose activity is strictly regulated by PKC. The aim of this study is to investigate the role of PKCα/GAP-43 nuclear signal transduction pathway during neuronal commitment of hPDLSCs. During hPDLSCs neurogenic commitment the levels of p-PKC and p-GAP-43 increased both in cytoplasmic and nuclear compartment. PKCα nuclear translocation induced GAP-43 movement to the cytoplasm, where it is known to regulate growth cone dynamics and neuronal differentiation. Moreover, the degree of cytosolic Ca(2+) mobilization appeared to be more pronounced in differentiated hPDLSCs than in undifferentiated cells. This study provides evidences of a new PKCα/GAP-43 nuclear signalling pathway that controls neuronal differentiation in hPDLSCs, leading the way to a potential use of these cells in cell-based therapy in neurodegenerative diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Domain-Specific Partitioning of Uterine Artery Endothelial Connexin43 and Caveolin-1.

PubMed

Ampey, Bryan C; Morschauser, Timothy J; Ramadoss, Jayanth; Magness, Ronald R

2016-10-01

Uterine vascular adaptations facilitate rises in uterine blood flow during pregnancy, which are associated with gap junction connexin (Cx) proteins and endothelial nitric oxide synthase. In uterine artery endothelial cells (UAECs), ATP activates endothelial nitric oxide synthase in a pregnancy (P)-specific manner that is dependent on Cx43 function. Caveolar subcellular domain partitioning plays key roles in ATP-induced endothelial nitric oxide synthase activation and nitric oxide production. Little is known regarding the partitioning of Cx proteins to caveolar domains or their dynamics with ATP treatment. We observed that Cx43-mediated gap junction function with ATP stimulation is associated with Cx43 repartitioning between the noncaveolar and caveolar domains. Compared with UAECs from nonpregnant (NP) ewes, levels of ATP, PGI2, cAMP, NOx, and cGMP were 2-fold higher (P<0.05) in pregnant UAECs. In pregnant UAECs, ATP increased Lucifer yellow dye transfer, a response abrogated by Gap27, but not Gap 26, indicating involvement of Cx43, but not Cx37. Confocal microscopy revealed domain partitioning of Cx43 and caveolin-1. In pregnant UAECs, LC/MS/MS analysis revealed only Cx43 in the caveolar domain. In contrast, Cx37 was located only in the noncaveolar pool. Western analysis revealed that ATP increased Cx43 distribution (1.7-fold; P=0.013) to the caveolar domain, but had no effect on Cx37. These data demonstrate rapid ATP-stimulated repartitioning of Cx43 to the caveolae, where endothelial nitric oxide synthase resides and plays an important role in nitric oxide-mediated increasing uterine blood flow during pregnancy. © 2016 American Heart Association, Inc.

Increased Cardiac Arrhythmogenesis Associated With Gap Junction Remodeling With Upregulation of RNA-Binding Protein FXR1.

PubMed

Chu, Miensheng; Novak, Stefanie Mares; Cover, Cathleen; Wang, Anne A; Chinyere, Ikeotunye Royal; Juneman, Elizabeth B; Zarnescu, Daniela C; Wong, Pak Kin; Gregorio, Carol C

2018-02-06

Gap junction remodeling is well established as a consistent feature of human heart disease involving spontaneous ventricular arrhythmia. The mechanisms responsible for gap junction remodeling that include alterations in the distribution of, and protein expression within, gap junctions are still debated. Studies reveal that multiple transcriptional and posttranscriptional regulatory pathways are triggered in response to cardiac disease, such as those involving RNA-binding proteins. The expression levels of FXR1 (fragile X mental retardation autosomal homolog 1), an RNA-binding protein, are critical to maintain proper cardiac muscle function; however, the connection between FXR1 and disease is not clear. To identify the mechanisms regulating gap junction remodeling in cardiac disease, we sought to identify the functional properties of FXR1 expression, direct targets of FXR1 in human left ventricle dilated cardiomyopathy (DCM) biopsy samples and mouse models of DCM through BioID proximity assay and RNA immunoprecipitation, how FXR1 regulates its targets through RNA stability and luciferase assays, and functional consequences of altering the levels of this important RNA-binding protein through the analysis of cardiac-specific FXR1 knockout mice and mice injected with 3xMyc-FXR1 adeno-associated virus. FXR1 expression is significantly increased in tissue samples from human and mouse models of DCM via Western blot analysis. FXR1 associates with intercalated discs, and integral gap junction proteins Cx43 (connexin 43), Cx45 (connexin 45), and ZO-1 (zonula occludens-1) were identified as novel mRNA targets of FXR1 by using a BioID proximity assay and RNA immunoprecipitation. Our findings show that FXR1 is a multifunctional protein involved in translational regulation and stabilization of its mRNA targets in heart muscle. In addition, introduction of 3xMyc-FXR1 via adeno-associated virus into mice leads to the redistribution of gap junctions and promotes ventricular tachycardia, showing the functional significance of FXR1 upregulation observed in DCM. In DCM, increased FXR1 expression appears to play an important role in disease progression by regulating gap junction remodeling. Together this study provides a novel function of FXR1, namely, that it directly regulates major gap junction components, contributing to proper cell-cell communication in the heart. © 2017 American Heart Association, Inc.

Prostacyclin primes pregnant human myometrium for an enhanced contractile response in parturition

PubMed Central

Fetalvero, Kristina M.; Zhang, Peisheng; Shyu, Maureen; Young, Benjamin T.; Hwa, John; Young, Roger C.; Martin, Kathleen A.

2008-01-01

An incomplete understanding of the molecular events that regulate the myometrial transition from the quiescent pregnant state to the active contractile state during labor has hindered the development of improved therapies for preterm labor. During myometrial activation, proteins that prime the smooth muscle for contraction are upregulated, allowing maximal responsiveness to contractile agonists and thereby producing strong phasic contractions. Upregulation of one such protein, COX-2, generates PGs that induce contractions. Intriguingly, the predominant myometrial PG produced just prior to labor is prostacyclin (PGI2), a smooth muscle relaxant. However, here we have shown that activation of PGI2 receptor (IP) upregulated the expression of several contractile proteins and the gap junction protein connexin 43 through cAMP/PKA signaling in human myometrial tissue in organ and cell culture. Functionally, these IP-dependent changes in gene expression promoted an enhanced contractile response to oxytocin in pregnant human myometrial tissue strips, which was inhibited by the IP antagonist RO3244794. Furthermore, contractile protein induction was dependent on the concentration and time of exposure to the PGI2 analog iloprost and was blocked by both RO3244794 and PKA knockdown. We therefore propose that PGI2-mediated upregulation of contractile proteins and connexin 43 is a critical step in myometrial activation, allowing for a maximal contractile response. Our observations have important implications regarding activation of the myometrium prior to the onset of labor. PMID:19033666

Drosophila Shaking-B protein forms gap junctions in paired Xenopus oocytes.

PubMed

Phelan, P; Stebbings, L A; Baines, R A; Bacon, J P; Davies, J A; Ford, C

1998-01-08

In most multicellular organisms direct cell-cell communication is mediated by the intercellular channels of gap junctions. These channels allow the exchange of ions and molecules that are believed to be essential for cell signalling during development and in some differentiated tissues. Proteins called connexins, which are products of a multigene family, are the structural components of vertebrate gap junctions. Surprisingly, molecular homologues of the connexins have not been described in any invertebrate. A separate gene family, which includes the Drosophila genes shaking-B and l(1)ogre, and the Caenorhabditis elegans genes unc-7 and eat-5, encodes transmembrane proteins with a predicted structure similar to that of the connexins. shaking-B and eat-5 are required for the formation of functional gap junctions. To test directly whether Shaking-B is a channel protein, we expressed it in paired Xenopus oocytes. Here we show that Shaking-B localizes to the membrane, and that its presence induces the formation of functional intercellular channels. To our knowledge, this is the first structural component of an invertebrate gap junction to be characterized.

Intracellular trafficking pathways of Cx43 gap junction channels.

PubMed

Epifantseva, Irina; Shaw, Robin M

2018-01-01

Gap Junction (GJ) channels, including the most common Connexin 43 (Cx43), have fundamental roles in excitable tissues by facilitating rapid transmission of action potentials between adjacent cells. For instance, synchronization during each heartbeat is regulated by these ion channels at the cardiomyocyte cell-cell border. Cx43 protein has a short half-life, and rapid synthesis and timely delivery of those proteins to particular subdomains are crucial for the cellular organization of gap junctions and maintenance of intracellular coupling. Impairment in gap junction trafficking contributes to dangerous complications in diseased hearts such as the arrhythmias of sudden cardiac death. Of recent interest are the protein-protein interactions with the Cx43 carboxy-terminus. These interactions have significant impact on the full length Cx43 lifecycle and also contribute to trafficking of Cx43 as well as possibly other functions. We are learning that many of the known non-canonical roles of Cx43 can be attributed to the recently identified six endogenous Cx43 truncated isoforms which are produced by internal translation. In general, alternative translation is a new leading edge for proteome expansion and therapeutic drug development. This review highlights recent mechanisms identified in the trafficking of gap junction channels, involvement of other proteins contributing to the delivery of channels to the cell-cell border, and understanding of possible roles of the newly discovered alternatively translated isoforms in Cx43 biology. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve. Copyright © 2017 Elsevier B.V. All rights reserved.

Non-Gaussian statistics and nanosecond dynamics of electrostatic fluctuations affecting optical transitions in proteins.

PubMed

Martin, Daniel R; Matyushov, Dmitry V

2012-08-30

We show that electrostatic fluctuations of the protein-water interface are globally non-Gaussian. The electrostatic component of the optical transition energy (energy gap) in a hydrated green fluorescent protein is studied here by classical molecular dynamics simulations. The distribution of the energy gap displays a high excess in the breadth of electrostatic fluctuations over the prediction of the Gaussian statistics. The energy gap dynamics include a nanosecond component. When simulations are repeated with frozen protein motions, the statistics shifts to the expectations of linear response and the slow dynamics disappear. We therefore suggest that both the non-Gaussian statistics and the nanosecond dynamics originate largely from global, low-frequency motions of the protein coupled to the interfacial water. The non-Gaussian statistics can be experimentally verified from the temperature dependence of the first two spectral moments measured at constant-volume conditions. Simulations at different temperatures are consistent with other indicators of the non-Gaussian statistics. In particular, the high-temperature part of the energy gap variance (second spectral moment) scales linearly with temperature and extrapolates to zero at a temperature characteristic of the protein glass transition. This result, violating the classical limit of the fluctuation-dissipation theorem, leads to a non-Boltzmann statistics of the energy gap and corresponding non-Arrhenius kinetics of radiationless electronic transitions, empirically described by the Vogel-Fulcher-Tammann law.

Structure of the catalytic domain of Plasmodium falciparum ARF GTPase-activating protein (ARFGAP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, William J.; Senkovich, Olga; Chattopadhyay, Debasish

2012-03-26

The crystal structure of the catalytic domain of the ADP ribosylation factor GTPase-activating protein (ARFGAP) from Plasmodium falciparum has been determined and refined to 2.4 {angstrom} resolution. Multiwavelength anomalous diffraction (MAD) data were collected utilizing the Zn{sup 2+} ion bound at the zinc-finger domain and were used to solve the structure. The overall structure of the domain is similar to those of mammalian ARFGAPs. However, several amino-acid residues in the area where GAP interacts with ARF1 differ in P. falciparum ARFGAP. Moreover, a number of residues that form the dimer interface in the crystal structure are unique in P. falciparummore » ARFGAP.« less

Optimal subset selection of primary sequence features using the genetic algorithm for thermophilic proteins identification.

PubMed

Wang, LiQiang; Li, CuiFeng

2014-10-01

A genetic algorithm (GA) coupled with multiple linear regression (MLR) was used to extract useful features from amino acids and g-gap dipeptides for distinguishing between thermophilic and non-thermophilic proteins. The method was trained by a benchmark dataset of 915 thermophilic and 793 non-thermophilic proteins. The method reached an overall accuracy of 95.4 % in a Jackknife test using nine amino acids, 38 0-gap dipeptides and 29 1-gap dipeptides. The accuracy as a function of protein size ranged between 85.8 and 96.9 %. The overall accuracies of three independent tests were 93, 93.4 and 91.8 %. The observed results of detecting thermophilic proteins suggest that the GA-MLR approach described herein should be a powerful method for selecting features that describe thermostabile machines and be an aid in the design of more stable proteins.

SCARFACE encodes an ARF-GAP that is required for normal auxin efflux and vein patterning in Arabidopsis.

PubMed

Sieburth, Leslie E; Muday, Gloria K; King, Edward J; Benton, Geoff; Kim, Sun; Metcalf, Kasee E; Meyers, Lindsay; Seamen, Emylie; Van Norman, Jaimie M

2006-06-01

To identify molecular mechanisms controlling vein patterns, we analyzed scarface (sfc) mutants. sfc cotyledon and leaf veins are largely fragmented, unlike the interconnected networks in wild-type plants. SFC encodes an ADP ribosylation factor GTPase activating protein (ARF-GAP), a class with well-established roles in vesicle trafficking regulation. Quadruple mutants of SCF and three homologs (ARF-GAP DOMAIN1, 2, and 4) showed a modestly enhanced vascular phenotype. Genetic interactions between sfc and pinoid and between sfc and gnom suggest a possible function for SFC in trafficking of auxin efflux regulators. Genetic analyses also revealed interaction with cotyledon vascular pattern2, suggesting that lipid-based signals may underlie some SFC ARF-GAP functions. To assess possible roles for SFC in auxin transport, we analyzed sfc roots, which showed exaggerated responses to exogenous auxin and higher auxin transport capacity. To determine whether PIN1 intracellular trafficking was affected, we analyzed PIN1:green fluorescent protein (GFP) dynamics using confocal microscopy in sfc roots. We found normal PIN1:GFP localization at the apical membrane of root cells, but treatment with brefeldin A resulted in PIN1 accumulating in smaller and more numerous compartments than in the wild type. These data suggest that SFC is required for normal intracellular transport of PIN1 from the plasma membrane to the endosome.

Interacting Network of the Gap Junction (GJ) Protein Connexin43 (Cx43) is Modulated by Ischemia and Reperfusion in the Heart.

PubMed

Martins-Marques, Tania; Anjo, Sandra Isabel; Pereira, Paulo; Manadas, Bruno; Girão, Henrique

2015-11-01

The coordinated and synchronized cardiac muscle contraction relies on an efficient gap junction-mediated intercellular communication (GJIC) between cardiomyocytes, which involves the rapid anisotropic impulse propagation through connexin (Cx)-containing channels, namely of Cx43, the most abundant Cx in the heart. Expectedly, disturbing mechanisms that affect channel activity, localization and turnover of Cx43 have been implicated in several cardiomyopathies, such as myocardial ischemia. Besides gap junction-mediated intercellular communication, Cx43 has been associated with channel-independent functions, including modulation of cell adhesion, differentiation, proliferation and gene transcription. It has been suggested that the role played by Cx43 is dictated by the nature of the proteins that interact with Cx43. Therefore, the characterization of the Cx43-interacting network and its dynamics is vital to understand not only the molecular mechanisms underlying pathological malfunction of gap junction-mediated intercellular communication, but also to unveil novel and unanticipated biological functions of Cx43. In the present report, we applied a quantitative SWATH-MS approach to characterize the Cx43 interactome in rat hearts subjected to ischemia and ischemia-reperfusion. Our results demonstrate that, in the heart, Cx43 interacts with proteins related with various biological processes such as metabolism, signaling and trafficking. The interaction of Cx43 with proteins involved in gene transcription strengthens the emerging concept that Cx43 has a role in gene expression regulation. Importantly, our data shows that the interactome of Cx43 (Connexome) is differentially modulated in diseased hearts. Overall, the characterization of Cx43-interacting network may contribute to the establishment of new therapeutic targets to modulate cardiac function in physiological and pathological conditions. Data are available via ProteomeXchange with identifier PXD002331. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Evidence That Differences in Fructosamine-3-Kinase Activity May Be Associated With the Glycation Gap in Human Diabetes.

PubMed

Dunmore, Simon J; Al-Derawi, Amr S; Nayak, Ananth U; Narshi, Aruna; Nevill, Alan M; Hellwig, Anne; Majebi, Andrew; Kirkham, Paul; Brown, James E; Singh, Baldev M

2018-01-01

The phenomenon of a discrepancy between glycated hemoglobin levels and other indicators of average glycemia may be due to many factors but can be measured as the glycation gap (GGap). This GGap is associated with differences in complications in patients with diabetes and may possibly be explained by dissimilarities in deglycation in turn leading to altered production of advanced glycation end products (AGEs). We hypothesized that variations in the level of the deglycating enzyme fructosamine-3-kinase (FN3K) might be associated with the GGap. We measured erythrocyte FN3K concentrations and enzyme activity in a population dichotomized for a large positive or negative GGap. FN3K protein was higher and we found a striking threefold greater activity (323%) at any given FN3K protein level in the erythrocytes of the negative-GGap group compared with the positive-GGap group. This was associated with lower AGE levels in the negative-GGap group (79%), lower proinflammatory adipokines (leptin-to-adiponectin ratio) (73%), and much lower prothrombotic PAI-1 levels (19%). We conclude that FN3K may play a key role in the GGap and thus diabetes complications such that FN3K may be a potential predictor of the risk of diabetes complications. Pharmacological modifications of its activity may provide a novel approach to their prevention. © 2017 by the American Diabetes Association.

Connexin composition in apposed gap junction hemiplaques revealed by matched double-replica freeze-fracture replica immunogold labeling.

PubMed

Rash, John E; Kamasawa, Naomi; Davidson, Kimberly G V; Yasumura, Thomas; Pereda, Alberto E; Nagy, James I

2012-06-01

Despite the combination of light-microscopic immunocytochemistry, histochemical mRNA detection techniques and protein reporter systems, progress in identifying the protein composition of neuronal versus glial gap junctions, determination of the differential localization of their constituent connexin proteins in two apposing membranes and understanding human neurological diseases caused by connexin mutations has been problematic due to ambiguities introduced in the cellular and subcellular assignment of connexins. Misassignments occurred primarily because membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques. Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica immunogold labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques. However, freeze-fracture replica immunogold labeling has been limited because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difficult to identify connexin coupling partners in hemiplaques removed by fracturing. We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins. This approach allows unambiguous identification of connexins and determination of the membrane "sidedness" and the identities of connexin coupling partners in homotypic and heterotypic gap junctions of vertebrate neurons.

Ultrastructure and regulation of lateralized connexin43 in the failing heart.

PubMed

Hesketh, Geoffrey G; Shah, Manish H; Halperin, Victoria L; Cooke, Carol A; Akar, Fadi G; Yen, Timothy E; Kass, David A; Machamer, Carolyn E; Van Eyk, Jennifer E; Tomaselli, Gordon F

2010-04-02

Gap junctions mediate cell-to-cell electric coupling of cardiomyocytes. The primary gap junction protein in the working myocardium, connexin43 (Cx43), exhibits increased localization at the lateral membranes of cardiomyocytes in a variety of heart diseases, although the precise location and function of this population is unknown. To define the subcellular location of lateralized gap junctions at the light and electron microscopic level, and further characterize the biochemical regulation of gap junction turnover. By electron microscopy, we characterized gap junctions formed between cardiomyocyte lateral membranes in failing canine ventricular myocardium. These gap junctions were varied in structure and appeared to be extensively internalizing. Internalized gap junctions were incorporated into multilamellar membrane structures, with features characteristic of autophagosomes. Intracellular Cx43 extensively colocalized with the autophagosome marker GFP-LC3 when both proteins were exogenously expressed in HeLa cells, and endogenous Cx43 colocalized with GFP-LC3 in neonatal rat ventricular myocytes. Furthermore, a distinct phosphorylated form of Cx43, as well as the autophagosome-targeted form of LC3 (microtubule-associated protein light chain 3) targeted to lipid rafts in cardiac tissue, and both were increased in heart failure. Our data demonstrate a previously unrecognized pathway of gap junction internalization and degradation in the heart and identify a cellular pathway with potential therapeutic implications.

StarD13 is a tumor suppressor in breast cancer that regulates cell motility and invasion

PubMed Central

HANNA, SAMER; KHALIL, BASSEM; NASRALLAH, ANITA; SAYKALI, BECHARA A.; SOBH, RANIA; NASSER, SELIM; EL-SIBAI, MIRVAT

2014-01-01

Breast cancer is one of the most commonly diagnosed cancers in women around the world. In general, the more aggressive the tumor, the more rapidly it grows and the more likely it metastasizes. Members of the Rho subfamily of small GTP-binding proteins (GTPases) play a central role in breast cancer cell motility and metastasis. The switch between active GTP-bound and inactive GDP-bound state is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and guanine-nucleotide dissociation inhibitors (GDIs). We studied the role of StarD13, a recently identified Rho-GAP that specifically inhibits the function of RhoA and Cdc42. We aimed to investigate its role in breast cancer proliferation and metastasis. The levels of expression of this Rho-GAP in tumor tissues of different grades were assayed using immunohistochemistry. We observed that, while the level of StarD13 expression decreases in cancer tissues compared to normal tissues, it increases as the grade of the tumor increased. This was consistent with the fact that although StarD13 was indeed a tumor suppressor in our breast cancer cells, as seen by its effect on cell proliferation, it was needed for cancer cell motility. In fact, StarD13 knockdown resulted in an inhibition of cell motility and cells were not able to detach their tail and move forward. Our study describes, for the first time, a tumor suppressor that plays a positive role in cancer motility. PMID:24627003

How Proteins Bind Macrocycles

PubMed Central

Villar, Elizabeth A.; Beglov, Dmitri; Chennamadhavuni, Spandan; Porco, John A.; Kozakov, Dima; Vajda, Sandor; Whitty, Adrian

2014-01-01

The potential utility of synthetic macrocycles as drugs, particularly against low druggability targets such as protein-protein interactions, has been widely discussed. There is little information, however, to guide the design of macrocycles for good target protein-binding activity or bioavailability. To address this knowledge gap we analyze the binding modes of a representative set of macrocycle-protein complexes. The results, combined with consideration of the physicochemical properties of approved macrocyclic drugs, allow us to propose specific guidelines for the design of synthetic macrocycles libraries possessing structural and physicochemical features likely to favor strong binding to protein targets and also good bioavailability. We additionally provide evidence that large, natural product derived macrocycles can bind to targets that are not druggable by conventional, drug-like compounds, supporting the notion that natural product inspired synthetic macrocycles can expand the number of proteins that are druggable by synthetic small molecules. PMID:25038790

Joint diseases: from connexins to gap junctions.

PubMed

Donahue, Henry J; Qu, Roy W; Genetos, Damian C

2017-12-19

Connexons form the basis of hemichannels and gap junctions. They are composed of six tetraspan proteins called connexins. Connexons can function as individual hemichannels, releasing cytosolic factors (such as ATP) into the pericellular environment. Alternatively, two hemichannel connexons from neighbouring cells can come together to form gap junctions, membrane-spanning channels that facilitate cell-cell communication by enabling signalling molecules of approximately 1 kDa to pass from one cell to an adjacent cell. Connexins are expressed in joint tissues including bone, cartilage, skeletal muscle and the synovium. Indicative of their importance as gap junction components, connexins are also known as gap junction proteins, but individual connexin proteins are gaining recognition for their channel-independent roles, which include scaffolding and signalling functions. Considerable evidence indicates that connexons contribute to the function of bone and muscle, but less is known about the function of connexons in other joint tissues. However, the implication that connexins and gap junctional channels might be involved in joint disease, including age-related bone loss, osteoarthritis and rheumatoid arthritis, emphasizes the need for further research into these areas and highlights the therapeutic potential of connexins.

Combined chemical and structural signals of biomaterials synergistically activate cell-cell communications for improving tissue regeneration.

PubMed

Xu, Yachen; Peng, Jinliang; Dong, Xin; Xu, Yuhong; Li, Haiyan; Chang, Jiang

2017-06-01

Biomaterials are only used as carriers of cells in the conventional tissue engineering. Considering the multi-cell environment and active cell-biomaterial interactions in tissue regeneration process, in this study, structural signals of aligned electrospun nanofibers and chemical signals of bioglass (BG) ionic products in cell culture medium are simultaneously applied to activate fibroblast-endothelial co-cultured cells in order to obtain an improved skin tissue engineering construct. Results demonstrate that the combined biomaterial signals synergistically activate fibroblast-endothelial co-culture skin tissue engineering constructs through promotion of paracrine effects and stimulation of gap junctional communication between cells, which results in enhanced vascularization and extracellular matrix protein synthesis in the constructs. Structural signals of aligned electrospun nanofibers play an important role in stimulating both of paracrine and gap junctional communication while chemical signals of BG ionic products mainly enhance paracrine effects. In vivo experiments reveal that the activated skin tissue engineering constructs significantly enhance wound healing as compared to control. This study indicates the advantages of synergistic effects between different bioactive signals of biomaterials can be taken to activate communication between different types of cells for obtaining tissue engineering constructs with improved functions. Tissue engineering can regenerate or replace tissue or organs through combining cells, biomaterials and growth factors. Normally, for repairing a specific tissue, only one type of cells, one kind of biomaterials, and specific growth factors are used to support cell growth. In this study, we proposed a novel tissue engineering approach by simply using co-cultured cells and combined biomaterial signals. Using a skin tissue engineering model, we successfully proved that the combined biomaterial signals such as surface nanostructures and bioactive ions could synergistically stimulate the cell-cell communication in co-culture system through paracrine effects and gap junction activation, and regulated expression of growth factors and extracellular matrix proteins, resulting in an activated tissue engineering constructs that significantly enhanced skin regeneration. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

CombAlign: a code for generating a one-to-many sequence alignment from a set of pairwise structure-based sequence alignments.

PubMed

Zhou, Carol L Ecale

2015-01-01

In order to better define regions of similarity among related protein structures, it is useful to identify the residue-residue correspondences among proteins. Few codes exist for constructing a one-to-many multiple sequence alignment derived from a set of structure or sequence alignments, and a need was evident for creating such a tool for combining pairwise structure alignments that would allow for insertion of gaps in the reference structure. This report describes a new Python code, CombAlign, which takes as input a set of pairwise sequence alignments (which may be structure based) and generates a one-to-many, gapped, multiple structure- or sequence-based sequence alignment (MSSA). The use and utility of CombAlign was demonstrated by generating gapped MSSAs using sets of pairwise structure-based sequence alignments between structure models of the matrix protein (VP40) and pre-small/secreted glycoprotein (sGP) of Reston Ebolavirus and the corresponding proteins of several other filoviruses. The gapped MSSAs revealed structure-based residue-residue correspondences, which enabled identification of structurally similar versus differing regions in the Reston proteins compared to each of the other corresponding proteins. CombAlign is a new Python code that generates a one-to-many, gapped, multiple structure- or sequence-based sequence alignment (MSSA) given a set of pairwise sequence alignments (which may be structure based). CombAlign has utility in assisting the user in distinguishing structurally conserved versus divergent regions on a reference protein structure relative to other closely related proteins. CombAlign was developed in Python 2.6, and the source code is available for download from the GitHub code repository.

GAP Final Technical Report 12-14-04

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andrew J. Bordner, PhD, Senior Research Scientist

2004-12-14

The Genomics Annotation Platform (GAP) was designed to develop new tools for high throughput functional annotation and characterization of protein sequences and structures resulting from genomics and structural proteomics, benchmarking and application of those tools. Furthermore, this platform integrated the genomic scale sequence and structural analysis and prediction tools with the advanced structure prediction and bioinformatics environment of ICM. The development of GAP was primarily oriented towards the annotation of new biomolecular structures using both structural and sequence data. Even though the amount of protein X-ray crystal data is growing exponentially, the volume of sequence data is growing even moremore » rapidly. This trend was exploited by leveraging the wealth of sequence data to provide functional annotation for protein structures. The additional information provided by GAP is expected to assist the majority of the commercial users of ICM, who are involved in drug discovery, in identifying promising drug targets as well in devising strategies for the rational design of therapeutics directed at the protein of interest. The GAP also provided valuable tools for biochemistry education, and structural genomics centers. In addition, GAP incorporates many novel prediction and analysis methods not available in other molecular modeling packages. This development led to signing the first Molsoft agreement in the structural genomics annotation area with the University of oxford Structural Genomics Center. This commercial agreement validated the Molsoft efforts under the GAP project and provided the basis for further development of the large scale functional annotation platform.« less

The Myosin IXb Motor Activity Targets the Myosin IXb RhoGAP Domain as Cargo to Sites of Actin Polymerization

PubMed Central

van den Boom, Frank; Düssmann, Heiko; Uhlenbrock, Katharina; Abouhamed, Marouan

2007-01-01

Myosin IXb (Myo9b) is a single-headed processive myosin that exhibits Rho GTPase-activating protein (RhoGAP) activity in its tail region. Using live cell imaging, we determined that Myo9b is recruited to extending lamellipodia, ruffles, and filopodia, the regions of active actin polymerization. A functional motor domain was both necessary and sufficient for targeting Myo9b to these regions. The head domains of class IX myosins comprise a large insertion in loop2. Deletion of the large Myo9b head loop 2 insertion abrogated the enrichment in extending lamellipodia and ruffles, but enhanced significantly the enrichment at the tips of filopodia and retraction fibers. The enrichment in the tips of filopodia and retraction fibers depended on four lysine residues C-terminal to the loop 2 insertion and the tail region. Fluorescence recovery after photobleaching and photoactivation experiments in lamellipodia revealed that the dynamics of Myo9b was comparable to that of actin. The exchange rates depended on the Myo9b motor region and motor activity, and they were also dependent on the turnover of F-actin. These results demonstrate that Myo9b functions as a motorized RhoGAP molecule in regions of actin polymerization and identify Myo9b head sequences important for in vivo motor properties. PMID:17314409

The effector and scaffolding proteins AF6 and MUPP1 interact with connexin36 and localize at gap junctions that form electrical synapses in rodent brain.

PubMed

Li, X; Lynn, B D; Nagy, J I

2012-01-01

Electrical synapses formed by neuronal gap junctions composed of connexin36 (Cx36) occur in most major structures in the mammalian central nervous system. These synapses link ensembles of neurons and influence their network properties. Little is known about the macromolecular constituents of neuronal gap junctions or how transmission through electrical synapses is regulated at the level of channel conductance or gap junction assembly/disassembly. Such knowledge is a prerequisite to understanding the roles of gap junctions in neuronal circuitry. Gap junctions share similarities with tight and adhesion junctions in that all three reside at close plasma membrane appositions, and therefore may associate with similar structural and regulatory proteins. Previously, we reported that the tight junction-associated protein zonula occludens-1 (ZO-1) interacts with Cx36 and is localized at gap junctions. Here, we demonstrate that two proteins known to be associated with tight and adherens junctions, namely AF6 and MUPP1, are components of neuronal gap junctions in rodent brain. By immunofluorescence, AF6 and MUPP1 were co-localized with Cx36 in many brain areas. Co-immunoprecipitation and pull-down approaches revealed an association of Cx36 with AF6 and MUPP1, which required the C-terminus PDZ domain interaction motif of Cx36 for interaction with the single PDZ domain of AF6 and with the 10th PDZ domain of MUPP1. As AF6 is a target of the cAMP/Epac/Rap1 signalling pathway and MUPP1 is a scaffolding protein that interacts with CaMKII, the present results suggest that AF6 may be a target for cAMP/Epac/Rap1 signalling at electrical synapses, and that MUPP1 may contribute to anchoring CaMKII at these synapses. © 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

New kids on the block: The Popeye domain containing (POPDC) protein family acting as a novel class of cAMP effector proteins in striated muscle.

PubMed

Brand, Thomas; Schindler, Roland

2017-12-01

The cyclic 3',5'-adenosine monophosphate (cAMP) signalling pathway constitutes an ancient signal transduction pathway present in prokaryotes and eukaryotes. Previously, it was thought that in eukaryotes three effector proteins mediate cAMP signalling, namely protein kinase A (PKA), exchange factor directly activated by cAMP (EPAC) and the cyclic-nucleotide gated channels. However, recently a novel family of cAMP effector proteins emerged and was termed the Popeye domain containing (POPDC) family, which consists of three members POPDC1, POPDC2 and POPDC3. POPDC proteins are transmembrane proteins, which are abundantly present in striated and smooth muscle cells. POPDC proteins bind cAMP with high affinity comparable to PKA. Presently, their biochemical activity is poorly understood. However, mutational analysis in animal models as well as the disease phenotype observed in patients carrying missense mutations suggests that POPDC proteins are acting by modulating membrane trafficking of interacting proteins. In this review, we will describe the current knowledge about this gene family and also outline the apparent gaps in our understanding of their role in cAMP signalling and beyond. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

PIP3-independent activation of TorC2 and PKB at the cell’s leading edge mediates chemotaxis

PubMed Central

Kamimura, Yoichiro; Xiong, Yuan; Iglesias, Pablo A.; Hoeller, Oliver; Bolourani, Parvin; Devreotes, Peter N.

2008-01-01

Summary Background Studies show that high phosphotidylinositol 3,4,5 tris phosphate (PIP3) promotes cytoskeletal rearrangements and alters cell motility and chemotaxis, possibly through activation of PKBs. However, chemotaxis can still occur in the absence of PIP3 and the identities of the PIP3 independent pathways remain unknown. Results Here, we outline a PIP3-independent pathway linking temporal and spatial activation of PKBs by Tor complex 2 (TorC2) to the chemotactic response. Within seconds of stimulating Dictyostelium cells with chemoattractant, two PKB homologs, PKBA and PKBR1, mediate transient phosphorylation of at least eight proteins, including Talin, PI4P 5-kinase, two RasGefs, and a RhoGap. Surprisingly, all of the substrates are phosphorylated with normal kinetics in cells lacking PI 3-kinase activity. Cells deficient in TorC2 or PKB activity show reduced phosphorylation of the endogenous substrates and are impaired in chemotaxis. The PKBs are activated through phosphorylation of their hydrophobic motifs via TorC2 and subsequent phosphorylation of their activation loops. These chemoattractant-inducible events restricted to the cell’s leading edge even in the absence of PIP3. Activation of TorC2 depends on heterotrimeric G-protein function and intermediate G-proteins, including Ras GTPases. Conclusions The data lead to a model where cytosolic TorC2, encountering locally activated small G-protein(s) at the leading of the cell, becomes activated and phosphorylates PKBs. These in turn phosphorylate a series of signaling and cytoskeletal proteins, thereby regulating directed migration. PMID:18635356

PAR-2, LGL-1 and the CDC-42 GAP CHIN-1 act in distinct pathways to maintain polarity in the C. elegans embryo

PubMed Central

Beatty, Alexander; Morton, Diane G.; Kemphues, Kenneth

2013-01-01

In the one-cell C. elegans embryo, polarity is maintained by mutual antagonism between the anterior cortical proteins PAR-3, PKC-3, PAR-6 and CDC-42, and the posterior cortical proteins PAR-2 and LGL-1 on the posterior cortex. The mechanisms by which these proteins interact to maintain polarity are incompletely understood. In this study, we investigate the interplay among PAR-2, LGL-1, myosin, the anterior PAR proteins and CDC-42. We find that PAR-2 and LGL-1 affect cortical myosin accumulation by different mechanisms. LGL-1 does not directly antagonize the accumulation of cortical myosin and instead plays a role in regulating PAR-6 levels. By contrast, PAR-2 likely has separate roles in regulating cortical myosin accumulation and preventing the expansion of the anterior cortical domain. We also provide evidence that asymmetry of active CDC-42 can be maintained independently of LGL-1 and PAR-2 by a redundant pathway that includes the CDC-42 GAP CHIN-1. Finally, we show that, in addition to its primary role in regulating the size of the anterior cortical domain via its binding to PAR-6, CDC-42 has a secondary role in regulating cortical myosin that is not dependent on PAR-6. PMID:23536568

PAR-2, LGL-1 and the CDC-42 GAP CHIN-1 act in distinct pathways to maintain polarity in the C. elegans embryo.

PubMed

Beatty, Alexander; Morton, Diane G; Kemphues, Kenneth

2013-05-01

In the one-cell C. elegans embryo, polarity is maintained by mutual antagonism between the anterior cortical proteins PAR-3, PKC-3, PAR-6 and CDC-42, and the posterior cortical proteins PAR-2 and LGL-1 on the posterior cortex. The mechanisms by which these proteins interact to maintain polarity are incompletely understood. In this study, we investigate the interplay among PAR-2, LGL-1, myosin, the anterior PAR proteins and CDC-42. We find that PAR-2 and LGL-1 affect cortical myosin accumulation by different mechanisms. LGL-1 does not directly antagonize the accumulation of cortical myosin and instead plays a role in regulating PAR-6 levels. By contrast, PAR-2 likely has separate roles in regulating cortical myosin accumulation and preventing the expansion of the anterior cortical domain. We also provide evidence that asymmetry of active CDC-42 can be maintained independently of LGL-1 and PAR-2 by a redundant pathway that includes the CDC-42 GAP CHIN-1. Finally, we show that, in addition to its primary role in regulating the size of the anterior cortical domain via its binding to PAR-6, CDC-42 has a secondary role in regulating cortical myosin that is not dependent on PAR-6.

Nanoscale manipulation of membrane curvature for probing endocytosis in live cells.

PubMed

Zhao, Wenting; Hanson, Lindsey; Lou, Hsin-Ya; Akamatsu, Matthew; Chowdary, Praveen D; Santoro, Francesca; Marks, Jessica R; Grassart, Alexandre; Drubin, David G; Cui, Yi; Cui, Bianxiao

2017-08-01

Clathrin-mediated endocytosis (CME) involves nanoscale bending and inward budding of the plasma membrane, by which cells regulate both the distribution of membrane proteins and the entry of extracellular species. Extensive studies have shown that CME proteins actively modulate the plasma membrane curvature. However, the reciprocal regulation of how the plasma membrane curvature affects the activities of endocytic proteins is much less explored, despite studies suggesting that membrane curvature itself can trigger biochemical reactions. This gap in our understanding is largely due to technical challenges in precisely controlling the membrane curvature in live cells. In this work, we use patterned nanostructures to generate well-defined membrane curvatures ranging from +50 nm to -500 nm radius of curvature. We find that the positively curved membranes are CME hotspots, and that key CME proteins, clathrin and dynamin, show a strong preference towards positive membrane curvatures with a radius <200 nm. Of ten CME-related proteins we examined, all show preferences for positively curved membrane. In contrast, other membrane-associated proteins and non-CME endocytic protein caveolin1 show no such curvature preference. Therefore, nanostructured substrates constitute a novel tool for investigating curvature-dependent processes in live cells.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu Shanshan; Zhang Hong; Matunis, Michael J.

SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMOmore » substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification.« less

Toll-like receptor signaling in vertebrates: testing the integration of protein, complex, and pathway data in the protein ontology framework.

PubMed

Arighi, Cecilia; Shamovsky, Veronica; Masci, Anna Maria; Ruttenberg, Alan; Smith, Barry; Natale, Darren A; Wu, Cathy; D'Eustachio, Peter

2015-01-01

The Protein Ontology (PRO) provides terms for and supports annotation of species-specific protein complexes in an ontology framework that relates them both to their components and to species-independent families of complexes. Comprehensive curation of experimentally known forms and annotations thereof is expected to expose discrepancies, differences, and gaps in our knowledge. We have annotated the early events of innate immune signaling mediated by Toll-Like Receptor 3 and 4 complexes in human, mouse, and chicken. The resulting ontology and annotation data set has allowed us to identify species-specific gaps in experimental data and possible functional differences between species, and to employ inferred structural and functional relationships to suggest plausible resolutions of these discrepancies and gaps.

The effectiveness of position- and composition-specific gap costs for protein similarity searches.

PubMed

Stojmirović, Aleksandar; Gertz, E Michael; Altschul, Stephen F; Yu, Yi-Kuo

2008-07-01

The flexibility in gap cost enjoyed by hidden Markov models (HMMs) is expected to afford them better retrieval accuracy than position-specific scoring matrices (PSSMs). We attempt to quantify the effect of more general gap parameters by separately examining the influence of position- and composition-specific gap scores, as well as by comparing the retrieval accuracy of the PSSMs constructed using an iterative procedure to that of the HMMs provided by Pfam and SUPERFAMILY, curated ensembles of multiple alignments. We found that position-specific gap penalties have an advantage over uniform gap costs. We did not explore optimizing distinct uniform gap costs for each query. For Pfam, PSSMs iteratively constructed from seeds based on HMM consensus sequences perform equivalently to HMMs that were adjusted to have constant gap transition probabilities, albeit with much greater variance. We observed no effect of composition-specific gap costs on retrieval performance. These results suggest possible improvements to the PSI-BLAST protein database search program. The scripts for performing evaluations are available upon request from the authors.

Heat shock protein 70 stimulation of the deoxyribonucleic acid base excision repair enzyme polymerase β

PubMed Central

Mendez, Frances; Kozin, Elliott; Bases, Robert

2003-01-01

Base excision repair (BER) of damaged deoxyribonucleic acid (DNA) is a multistep process during which potentially lethal abasic sites temporarily exist. Repair of these lesions is greatly stimulated by heat shock protein 70 (Hsp70), which enhances strand incision and removal of the abasic sites by human apurinic-apyrimidinic endonuclease (HAP1). The resulting single-strand gaps must then be filled in. Here, we show that Hsp70 and its 48- and 43-kDa N-terminal domains greatly stimulated filling in the single-strand gaps by DNA polymerase β, a novel finding that extends the role of Hsps in DNA repair. Incorporation of deoxyguanosine monophosphate (dGMP) to fill in single-strand gaps in DNA phagemid pBKS by DNA polymerase β was stimulated by Hsp70. Truncated proteins derived from the C-terminus of Hsp70 as well as unrelated proteins were less effective, but proteins derived from the N-terminus of Hsp70 remained efficient stimulators of DNA polymerase β repair of DNA single-strand gaps. In agreement with these results, repair of a gap in a 30-bp oligonucleotide by polymerase β also was strongly stimulated by Hsp70 although not by a truncated protein from the C-terminus of Hsp70. Sealing of the repaired site in the oligonucleotide by human DNA ligase 1 was not specifically stimulated by Hsp-related proteins. Results presented here now implicate and extend the role of Hsp70 as a partner in the enzymatic repair of damaged DNA. The participation of Hsp70 jointly with base excision enzymes improves repair efficiency by mechanisms that are not yet understood. PMID:14627201

Dragging ras back in the ring.

PubMed

Stephen, Andrew G; Esposito, Dominic; Bagni, Rachel K; McCormick, Frank

2014-03-17

Ras proteins play a major role in human cancers but have not yielded to therapeutic attack. Ras-driven cancers are among the most difficult to treat and often excluded from therapies. The Ras proteins have been termed "undruggable," based on failures from an era in which understanding of signaling transduction, feedback loops, redundancy, tumor heterogeneity, and Ras' oncogenic role was poor. Structures of Ras oncoproteins bound to their effectors or regulators are unsolved, and it is unknown precisely how Ras proteins activate their downstream targets. These knowledge gaps have impaired development of therapeutic strategies. A better understanding of Ras biology and biochemistry, coupled with new ways of targeting undruggable proteins, is likely to lead to new ways of defeating Ras-driven cancers. Copyright © 2014 Elsevier Inc. All rights reserved.

G proteins as regulators in ethylene-mediated hypoxia signaling

PubMed Central

Sauter, Margret

2010-01-01

Waterlogging or flooding are frequently or constitutively encountered by many plant species. The resulting reduction in endogenous O2 concentration poses a severe threat. Numerous adaptations at the anatomical, morphological and metabolic level help plants to either escape low oxygen conditions or to endure them. Formation of aerenchyma or rapid shoot elongation are escape responses, as is the formation of adventitious roots. The metabolic shift from aerobic respiration to anaerobic fermentation contributes to a basal energy supply at low oxygen conditions. Ethylene plays a central role in hypoxic stress signaling, and G proteins have been recognized as crucial signal transducers in various hypoxic signaling pathways. The programmed death of parenchyma cells that results in hypoxia-induced aerenchyma formation is an ethylene response. In maize, aerenchyma are induced in the absence of ethylene when G proteins are constitutively activated. Similarly, ethylene induced death of epidermal cells that cover adventitious roots at the stem node of rice is strictly dependent on heterotrimeric G protein activity. Knock down of the unique Gα gene RGA1 in rice prevents epidermal cell death. Finally, in Arabidopsis, induction of alcohol dehydrogenase with resulting increased plant survival relies on the balanced activities of a small Rop G protein and its deactivating protein RopGAP4. Identifying the general mechanisms of G protein signaling in hypoxia adaptation of plants is one of the tasks ahead. PMID:20948297

Expression of gap junction protein connexin 43 in bovine urinary bladder tumours.

PubMed

Corteggio, A; Florio, J; Roperto, F; Borzacchiello, G

2011-01-01

The aetiopathogenesis of urinary bladder tumours in cattle involves prolonged ingestion of bracken fern and infection by bovine papillomavirus types 1 or 2 (BPV-1/2). The oncogenic activity of BPV is largely associated with the major oncoprotein E5. Gap junctions are the only communicating junctions found in animal tissues and are composed of proteins known as connexins. Alterations in connexin expression have been associated with oncogenesis. The present study investigated biochemically and immunohistochemically the expression of connexin 43 in samples of normal (n=2), dysplastic (n=3) and neoplastic (n=23) bovine urothelium. The tumours included 10 carcinomas in situ, five papillary urothelial carcinomas and eight invasive urothelial carcinomas. Normal and dysplastic urothelium had membrane expression of connexin 43, but this was reduced in samples of carcinoma in situ. Papillary urothelial carcinomas showed moderate cytoplasmic and membrane labelling, while invasive carcinoma showed loss of connexin 43 expression. Copyright © 2010 Elsevier Ltd. All rights reserved.

All known patient mutations in the ASH-RhoGAP domains of OCRL affect targeting and APPL1 binding

PubMed Central

McCrea, Heather J.; Paradise, Summer; Tomasini, Livia; Addis, Maria; Melis, Maria Antonietta; De Matteis, Maria Antonietta; De Camilli, Pietro

2008-01-01

Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome, an X-linked disorder characterized by bilateral cataracts, mental retardation, neonatal hypotonia, and renal Fanconi syndrome, and for Dent disease, another X-linked condition characterized by kidney reabsorption defects. We have previously described an interaction of OCRL with the endocytic adaptor APPL1 that links OCRL to protein networks involved in the disease phenotype. Here we provide new evidence showing that among the interactions which target OCRL to membranes of the endocytic pathway, binding to APPL1 is the only one abolished by all known disease-causing missense mutations in the ASH-RhoGAP domains of the protein. Furthermore, we demonstrate that APPL1 and rab5 independently contribute to recruit OCRL to enlarged endosomes induced by the expression of constitutively active Rab5. Thus, binding to APPL1 helps localize OCRL at specific cellular sites, and disruption of this interaction may play a role in disease. PMID:18307981

Connexin 43 and ATP-sensitive potassium channels crosstalk: a missing link in hypoxia/ischemia stress.

PubMed

Ahmad Waza, Ajaz; Ahmad Bhat, Shabir; Ul Hussain, Mahboob; Ganai, Bashir A

2018-02-01

Connexin 43 (Cx43) is a gap junction protein expressed in various tissues and organs of vertebrates. Besides functioning as a gap junction, Cx43 also regulates diverse cellular processes like cell growth and differentiation, cell migration, cell survival, etc. Cx43 is critical for normal cardiac functioning and is therefore abundantly expressed in cardiomyocytes. On the other hand, ATP-sensitive potassium (K ATP ) channels are metabolic sensors converting metabolic changes into electrical activity. These channels are important in maintaining the neurotransmitter release, smooth muscle relaxation, cardiac action potential repolarization, normal physiology of cellular repolarization, insulin secretion and immune function. Cx43 and K ATP channels are part of the same signaling pathway, regulating cell survival during stress conditions and ischemia/hypoxia preconditioning. However, the underlying molecular mechanism for their combined role in ischemia/hypoxia preconditioning is largely unknown. The current review focuses on understanding the molecular mechanism responsible for the coordinated role of Cx43 and K ATP channel protein in protecting cardiomyocytes against ischemia/hypoxia stress.

TC-PTP directly interacts with connexin43 to regulate gap junction intercellular communication

PubMed Central

Li, Hanjun; Spagnol, Gaelle; Naslavsky, Naava; Caplan, Steve; Sorgen, Paul L.

2014-01-01

ABSTRACT Protein kinases have long been reported to regulate connexins; however, little is known about the involvement of phosphatases in the modulation of intercellular communication through gap junctions and the subsequent downstream effects on cellular processes. Here, we identify an interaction between the T-cell protein tyrosine phosphatase (TC-PTP, officially known as PTPN2) and the carboxyl terminus of connexin43 (Cx43, officially known as GJA1). Two cell lines, normal rat kidney (NRK) cells endogenously expressing Cx43 and an NRK-derived cell line expressing v-Src with temperature-sensitive activity, were used to demonstrate that EGF and v-Src stimulation, respectively, induced TC-PTP to colocalize with Cx43 at the plasma membrane. Cell biology experiments using phospho-specific antibodies and biophysical assays demonstrated that the interaction is direct and that TC-PTP dephosphorylates Cx43 residues Y247 and Y265, but does not affect v-Src. Transfection of TC-PTP also indirectly led to the dephosphorylation of Cx43 S368, by inactivating PKCα and PKCδ, with no effect on the phosphorylation of S279 and S282 (MAPK-dependent phosphorylation sites). Dephosphorylation maintained Cx43 gap junctions at the plaque and partially reversed the channel closure caused by v-Src-mediated phosphorylation of Cx43. Understanding dephosphorylation, along with the well-documented roles of Cx43 phosphorylation, might eventually lead to methods to modulate the regulation of gap junction channels, with potential benefits for human health. PMID:24849651

Gap Junction Proteins in the Blood-Brain Barrier Control Nutrient-Dependent Reactivation of Drosophila Neural Stem Cells

PubMed Central

Spéder, Pauline; Brand, Andrea H.

2014-01-01

Summary Neural stem cells in the adult brain exist primarily in a quiescent state but are reactivated in response to changing physiological conditions. How do stem cells sense and respond to metabolic changes? In the Drosophila CNS, quiescent neural stem cells are reactivated synchronously in response to a nutritional stimulus. Feeding triggers insulin production by blood-brain barrier glial cells, activating the insulin/insulin-like growth factor pathway in underlying neural stem cells and stimulating their growth and proliferation. Here we show that gap junctions in the blood-brain barrier glia mediate the influence of metabolic changes on stem cell behavior, enabling glia to respond to nutritional signals and reactivate quiescent stem cells. We propose that gap junctions in the blood-brain barrier are required to translate metabolic signals into synchronized calcium pulses and insulin secretion. PMID:25065772

Iron oxide nanoparticle-mediated development of cellular gap junction crosstalk to improve mesenchymal stem cells' therapeutic efficacy for myocardial infarction.

PubMed

Han, Jin; Kim, Bokyoung; Shin, Jung-Youn; Ryu, Seungmi; Noh, Myungkyung; Woo, Jongsu; Park, Jin-Sil; Lee, Youjin; Lee, Nohyun; Hyeon, Taeghwan; Choi, Donghoon; Kim, Byung-Soo

2015-03-24

Electrophysiological phenotype development and paracrine action of mesenchymal stem cells (MSCs) are the critical factors that determine the therapeutic efficacy of MSCs for myocardial infarction (MI). In such respect, coculture of MSCs with cardiac cells has windowed a platform for cardiac priming of MSCs. Particularly, active gap junctional crosstalk of MSCs with cardiac cells in coculture has been known to play a major role in the MSC modification through coculture. Here, we report that iron oxide nanoparticles (IONPs) significantly augment the expression of connexin 43 (Cx43), a gap junction protein, of cardiomyoblasts (H9C2), which would be critical for gap junctional communication with MSCs in coculture for the generation of therapeutic potential-improved MSCs. MSCs cocultured with IONP-harboring H9C2 (cocultured MSCs: cMSCs) showed active cellular crosstalk with H9C2 and displayed significantly higher levels of electrophysiological cardiac biomarkers and a cardiac repair-favorable paracrine profile, both of which are responsible for MI repair. Accordingly, significantly improved animal survival and heart function were observed upon cMSC injection into rat MI models compared with the injection of unmodified MSCs. The present study highlights an application of IONPs in developing gap junctional crosstalk among the cells and generating cMSCs that exceeds the reparative potentials of conventional MSCs. On the basis of our finding, the potential application of IONPs can be extended in cell biology and stem cell-based therapies.

TBC-8, a putative RAB-2 GAP, regulates dense core vesicle maturation in Caenorhabditis elegans.

PubMed

Hannemann, Mandy; Sasidharan, Nikhil; Hegermann, Jan; Kutscher, Lena M; Koenig, Sabine; Eimer, Stefan

2012-01-01

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.

Low Level Pro-inflammatory Cytokines Decrease Connexin36 Gap Junction Coupling in Mouse and Human Islets through Nitric Oxide-mediated Protein Kinase Cδ*

PubMed Central

Farnsworth, Nikki L.; Walter, Rachelle L.; Hemmati, Alireza; Westacott, Matthew J.; Benninger, Richard K. P.

2016-01-01

Pro-inflammatory cytokines contribute to the decline in islet function during the development of diabetes. Cytokines can disrupt insulin secretion and calcium dynamics; however, the mechanisms underlying this are poorly understood. Connexin36 gap junctions coordinate glucose-induced calcium oscillations and pulsatile insulin secretion across the islet. Loss of gap junction coupling disrupts these dynamics, similar to that observed during the development of diabetes. This study investigates the mechanisms by which pro-inflammatory cytokines mediate gap junction coupling. Specifically, as cytokine-induced NO can activate PKCδ, we aimed to understand the role of PKCδ in modulating cytokine-induced changes in gap junction coupling. Isolated mouse and human islets were treated with varying levels of a cytokine mixture containing TNF-α, IL-1β, and IFN-γ. Islet dysfunction was measured by insulin secretion, calcium dynamics, and gap junction coupling. Modulators of PKCδ and NO were applied to determine their respective roles in modulating gap junction coupling. High levels of cytokines caused cell death and decreased insulin secretion. Low levels of cytokine treatment disrupted calcium dynamics and decreased gap junction coupling, in the absence of disruptions to insulin secretion. Decreases in gap junction coupling were dependent on NO-regulated PKCδ, and altered membrane organization of connexin36. This study defines several mechanisms underlying the disruption to gap junction coupling under conditions associated with the development of diabetes. These mechanisms will allow for greater understanding of islet dysfunction and suggest ways to ameliorate this dysfunction during the development of diabetes. PMID:26668311

The Arf GEF GBF1 and Arf4 synergize with the sensory receptor cargo, rhodopsin, to regulate ciliary membrane trafficking.

PubMed

Wang, Jing; Fresquez, Theresa; Kandachar, Vasundhara; Deretic, Dusanka

2017-12-01

The small GTPase Arf4 and the Arf GTPase-activating protein (GAP) ASAP1 cooperatively sequester sensory receptor cargo into transport carriers targeted to primary cilia, but the input that drives Arf4 activation in this process remains unknown. Here, we show, by using frog retinas and recombinant human proteins, that during the carrier biogenesis from the photoreceptor Golgi/ trans -Golgi network (TGN) a functional complex is formed between Arf4, the Arf guanine nucleotide exchange factor (GEF) GBF1 and the light-sensing receptor, rhodopsin. Rhodopsin and Arf4 bind the regulatory N-terminal dimerization and cyclophillin-binding (DCB)-homology upstream of Sec7 (HUS) domain of GBF1. The complex is sensitive to Golgicide A (GCA), a selective inhibitor of GBF1 that accordingly blocks rhodopsin delivery to the cilia, without disrupting the photoreceptor Golgi. The emergence of newly synthesized rhodopsin in the endomembrane system is essential for GBF1-Arf4 complex formation in vivo Notably, GBF1 interacts with the Arf GAP ASAP1 in a GCA-resistant manner. Our findings indicate that converging signals on GBF1 from the influx of cargo into the Golgi/TGN and the feedback from Arf4, combined with input from ASAP1, control Arf4 activation during sensory membrane trafficking to primary cilia. © 2017. Published by The Company of Biologists Ltd.

Gap-Junctional communication between developing Drosophila muscles is essential for their normal development.

PubMed

Todman, M G; Baines, R A; Stebbings, L A; Davies, J A; Bacon, J P

1999-01-01

Recent experiments have demonstrated that a family of proteins, known as the innexins, are structural components of invertebrate gap junctions. The shaking-B (shak-B) locus of Drosophila encodes two members of this emerging family, Shak-B(lethal) and Shak-B(neural). This study focuses on the role of Shak-B gap junctions in the development of embryonic and larval muscle. During embryogenesis, shak-B transcripts are expressed in a subset of the somatic muscles; expression is strong in ventral oblique muscles (VO4-6) but only weak in ventral longitudinals (VL3 and 4). Carboxyfluorescein injected into VO4 of wild-type early stage 16 embryos spreads, via gap junctions, to label adjacent muscles, including VL3 and 4. In shak-B2 embryos (in which the shak-B(neural) function is disrupted), dye injected into VO4 fails to spread into other muscles. In the first instar larva, when dye coupling between muscles is no longer present, another effect of the shak-B2 mutation is revealed by whole-cell voltage clamp. In a calcium-free saline, only two voltage-activated potassium currents are present in wild-type muscles; a fast IA and a slow IK current. In shak-B2 larvae, these two currents are significantly reduced in magnitude in VO4 and 5, but remain normal in VL3. Expression of shak-B(neural) in a shak-B2 background fully rescues both dye coupling in embryonic muscle and whole-cell currents in first instar VO4 and 5. Our observations show that Shak-B(neural) is one of a set of embryonic gap-junction proteins, and that it is required for the normal temporal development of potassium currents in some larval muscles.

Potential Activities of Freshwater Exo- and Endo-Acting Extracellular Peptidases in East Tennessee and the Pocono Mountains.

PubMed

Mullen, Lauren; Boerrigter, Kim; Ferriero, Nicholas; Rosalsky, Jeff; Barrett, Abigail van Buren; Murray, Patrick J; Steen, Andrew D

2018-01-01

Proteins constitute a particularly bioavailable subset of organic carbon and nitrogen in aquatic environments but must be hydrolyzed by extracellular enzymes prior to being metabolized by microorganisms. Activities of extracellular peptidases (protein-degrading enzymes) have frequently been assayed in freshwater systems, but such studies have been limited to substrates for a single enzyme [leucyl aminopeptidase (Leu-AP)] out of more than 300 biochemically recognized peptidases. Here, we report kinetic measurements of extracellular hydrolysis of five substrates in 28 freshwater bodies in the Delaware Water Gap National Recreation Area in the Pocono Mountains (PA, United States) and near Knoxville (TN, United States), between 2013 and 2016. The assays putatively test for four aminopeptidases (arginyl aminopeptidase, glyclyl aminopeptidase, Leu-AP, and pyroglutamyl aminopeptidase), which cleave N -terminal amino acids from proteins, and trypsin, an endopeptidase, which cleaves proteins mid-chain. Aminopeptidase and the trypsin-like activity were observed in all water bodies, indicating that a diverse set of peptidases is typical in freshwater. However, ratios of peptidase activities were variable among sites: aminopeptidases dominated at some sites and trypsin-like activity at others. At a given site, the ratios remained fairly consistent over time, indicating that they are driven by ecological factors. Studies in which only Leu-AP activity is measured may underestimate the total peptidolytic capacity of an environment, due to the variable contribution of endopeptidases.

Potential Activities of Freshwater Exo- and Endo-Acting Extracellular Peptidases in East Tennessee and the Pocono Mountains

PubMed Central

Mullen, Lauren; Boerrigter, Kim; Ferriero, Nicholas; Rosalsky, Jeff; Barrett, Abigail van Buren; Murray, Patrick J.; Steen, Andrew D.

2018-01-01

Proteins constitute a particularly bioavailable subset of organic carbon and nitrogen in aquatic environments but must be hydrolyzed by extracellular enzymes prior to being metabolized by microorganisms. Activities of extracellular peptidases (protein-degrading enzymes) have frequently been assayed in freshwater systems, but such studies have been limited to substrates for a single enzyme [leucyl aminopeptidase (Leu-AP)] out of more than 300 biochemically recognized peptidases. Here, we report kinetic measurements of extracellular hydrolysis of five substrates in 28 freshwater bodies in the Delaware Water Gap National Recreation Area in the Pocono Mountains (PA, United States) and near Knoxville (TN, United States), between 2013 and 2016. The assays putatively test for four aminopeptidases (arginyl aminopeptidase, glyclyl aminopeptidase, Leu-AP, and pyroglutamyl aminopeptidase), which cleave N-terminal amino acids from proteins, and trypsin, an endopeptidase, which cleaves proteins mid-chain. Aminopeptidase and the trypsin-like activity were observed in all water bodies, indicating that a diverse set of peptidases is typical in freshwater. However, ratios of peptidase activities were variable among sites: aminopeptidases dominated at some sites and trypsin-like activity at others. At a given site, the ratios remained fairly consistent over time, indicating that they are driven by ecological factors. Studies in which only Leu-AP activity is measured may underestimate the total peptidolytic capacity of an environment, due to the variable contribution of endopeptidases. PMID:29559961

Role of connexins in metastatic breast cancer and melanoma brain colonization

PubMed Central

Stoletov, Konstantin; Strnadel, Jan; Zardouzian, Erin; Momiyama, Masashi; Park, Frederick D.; Kelber, Jonathan A.; Pizzo, Donald P.; Hoffman, Robert; VandenBerg, Scott R.; Klemke, Richard L.

2013-01-01

Summary Breast cancer and melanoma cells commonly metastasize to the brain using homing mechanisms that are poorly understood. Cancer patients with brain metastases display poor prognosis and survival due to the lack of effective therapeutics and treatment strategies. Recent work using intravital microscopy and preclinical animal models indicates that metastatic cells colonize the brain, specifically in close contact with the existing brain vasculature. However, it is not known how contact with the vascular niche promotes microtumor formation. Here, we investigate the role of connexins in mediating early events in brain colonization using transparent zebrafish and chicken embryo models of brain metastasis. We provide evidence that breast cancer and melanoma cells utilize connexin gap junction proteins (Cx43, Cx26) to initiate brain metastatic lesion formation in association with the vasculature. RNAi depletion of connexins or pharmacological blocking of connexin-mediated cell–cell communication with carbenoxolone inhibited brain colonization by blocking tumor cell extravasation and blood vessel co-option. Activation of the metastatic gene twist in breast cancer cells increased Cx43 protein expression and gap junction communication, leading to increased extravasation, blood vessel co-option and brain colonization. Conversely, inhibiting twist activity reduced Cx43-mediated gap junction coupling and brain colonization. Database analyses of patient histories revealed increased expression of Cx26 and Cx43 in primary melanoma and breast cancer tumors, respectively, which correlated with increased cancer recurrence and metastasis. Together, our data indicate that Cx43 and Cx26 mediate cancer cell metastasis to the brain and suggest that connexins might be exploited therapeutically to benefit cancer patients with metastatic disease. PMID:23321642

Rasfonin, a novel 2-pyrone derivative, induces ras-mutated Panc-1 pancreatic tumor cell death in nude mice.

PubMed

Xiao, Z; Li, L; Li, Y; Zhou, W; Cheng, J; Liu, F; Zheng, P; Zhang, Y; Che, Y

2014-05-22

Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR-Ras-Raf-MEK-ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [(3)H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC50=5.5 μM) than BxPC-3 cells (IC50=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras-MAPK activity could be important in its anticancer activity.

High-order oligomers of intrinsically disordered brain proteins BASP1 and GAP-43 preserve the structural disorder.

PubMed

Forsova, Oksana S; Zakharov, Vladislav V

2016-04-01

Brain acid-soluble protein-1 (BASP1) and growth-associated protein-43 (GAP-43) are presynaptic membrane proteins participating in axon guidance, neuroregeneration and synaptic plasticity. They are presumed to sequester phosphatidylinositol-4,5-bisphosphate (PIP2 ) in lipid rafts. Previously we have shown that the proteins form heterogeneously sized oligomers in the presence of anionic phospholipids or SDS at submicellar concentration. BASP1 and GAP-43 are intrinsically disordered proteins (IDPs). In light of this, we investigated the structure of their oligomers. Using partial cross-linking of the oligomers with glutaraldehyde, the aggregation numbers of BASP1 and GAP-43 were estimated as 10-14 and 6-7 monomer subunits, respectively. The cross-linking pattern indicated that the subunits are circularly arranged. The circular dichroism (CD) spectra of the monomers were characteristic of coil-like IDPs showing unordered structure with a high population of polyproline-II conformation. The oligomerization was accompanied by a minor CD spectral change attributable to formation of a small amount of α-helix. The number of residues in the α-helical conformation was estimated as 13 in BASP1 and 18 in GAP-43. However, the overall structure of the oligomers remained disordered, indicating a high degree of 'fuzziness'. This was confirmed by measuring the hydrodynamic dimensions of the oligomers using polyacrylamide gradient gel electrophoresis and size-exclusion chromatography, and by assaying their sensitivity to proteolytic digestion. There is evidence that the observed α-helical folding occurs within the basic effector domains, which are presumably tethered together via anionic molecules of SDS or PIP2 . We conclude that BASP1 and GAP-43 oligomers preserve a mostly disordered structure, which may be of great importance for their function in PIP2 signaling pathway. © 2016 Federation of European Biochemical Societies.

Long term effects of lipopolysaccharide on satellite glial cells in mouse dorsal root ganglia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blum, E.; Procacci, P.; Conte, V.

Lipopolysaccharide (LPS) has been used extensively to study neuroinflammation, but usually its effects were examined acutely (24 h<). We have shown previously that a single intraperitoneal LPS injection activated satellite glial cells (SGCs) in mouse dorsal root ganglia (DRG) and altered several functional parameters in these cells for at least one week. Here we asked whether the LPS effects would persist for 1 month. We injected mice with a single LPS dose and tested pain behavior, assessed SGCs activation in DRG using glial fibrillary acidic protein (GFAP) immunostaining, and injected a fluorescent dye intracellularly to study intercellular coupling. Electron microscopymore » was used to quantitate changes in gap junctions. We found that at 30 days post-LPS the threshold to mechanical stimulation was lower than in controls. GFAP expression, as well as the magnitude of dye coupling among SGCs were greater than in controls. Electron microscopy analysis supported these results, showing a greater number of gap junctions and an abnormal growth of SGC processes. These changes were significant, but less prominent than at 7 days post-LPS. We conclude that a single LPS injection exerts long-term behavioral and cellular changes. The results are consistent with the idea that SGC activation contributes to hyperalgesia. - Highlights: • A single lipopolysaccharides injection activated glia in mouse dorsal root ganglia for 30 days. • This was accompanied by increased communications by gap junctions among glia and by hyperalgesia. • Glial activation and coupling may contribute to chronic pain.« less

Phorbol ester and hydrogen peroxide synergistically induce the interaction of diacylglycerol kinase gamma with the Src homology 2 and C1 domains of beta2-chimaerin.

PubMed

Yasuda, Satoshi; Kai, Masahiro; Imai, Shin-ichi; Kanoh, Hideo; Sakane, Fumio

2008-01-01

DGKgamma (diacylglycerol kinase gamma) was reported to interact with beta2-chimaerin, a GAP (GTPase-activating protein) for Rac, in response to epidermal growth factor. Here we found that PMA and H2O2 also induced the interaction of DGKgamma with beta2-chimaerin. It is noteworthy that simultaneous addition of PMA and H2O2 synergistically enhanced the interaction. In this case, PMA was replaceable by DAG (diacylglycerol). The beta2-chimaerin translocation from the cytoplasm to the plasma membrane caused by PMA plus H2O2 was further enhanced by the expression of DGKgamma. Moreover, DGKgamma apparently enhanced the beta2-chimaerin GAP activity upon cell stimulation with PMA. PMA was found to be mainly required for a conversion of beta2-chimaerin into an active form. On the other hand, H2O2 was suggested to induce a release of Zn2+ from the C1 domain of beta2-chimaerin. By stepwise deletion analysis, we demonstrated that the SH2 (Src homology 2) and C1 domains of beta2-chimaerin interacted with the N-terminal half of catalytic region of DGKgamma. Unexpectedly, the SH2 domain of beta2-chimaerin contributes to the interaction independently of phosphotyrosine. Taken together, these results suggest that the functional link between DGKgamma and beta2-chimaerin has a broad significance in response to a wide range of cell stimuli. Our work offers a novel mechanism of protein-protein interaction, that is, the phosphotyrosine-independent interaction of the SH2 domain acting in co-operation with the C1 domain.

Structural plasticity mediates distinct GAP-dependent GTP hydrolysis mechanisms in Rab33 and Rab5.

PubMed

Majumdar, Soneya; Acharya, Abhishek; Prakash, Balaji

2017-12-01

The classical GTP hydrolysis mechanism, as seen in Ras, employs a catalytic glutamine provided in cis by the GTPase and an arginine supplied in trans by a GTPase activating protein (GAP). The key idea emergent from a large body of research on small GTPases is that GTPases employ a variety of different hydrolysis mechanisms; evidently, these variations permit diverse rates of GTPase inactivation, crucial for temporal regulation of different biological processes. Recently, we unified these variations and argued that a steric clash between active site residues (corresponding to positions 12 and 61 of Ras) governs whether a GTPase utilizes the cis-Gln or the trans-Gln (from the GAP) for catalysis. As the cis-Gln encounters a steric clash, the Rab GTPases employ the so-called dual finger mechanism where the interacting GAP supplies a trans-Gln for catalysis. Using experimental and computational methods, we demonstrate how the cis-Gln of Rab33 overcomes the steric clash when it is stabilized by a residue in the vicinity. In effect, this demonstrates how both cis-Gln- and trans-Gln-mediated mechanisms could operate in the same GTPase in different contexts, i.e. depending on the GAP that regulates its action. Interestingly, in the case of Rab5, which possesses a higher intrinsic GTP hydrolysis rate, a similar stabilization of the cis-Gln appears to overcome the steric clash. Taken together with the mechanisms seen for Rab1, it is evident that the observed variations in Rab and their GAP partners allow structural plasticity, or in other words, the choice of different catalytic mechanisms. © 2017 Federation of European Biochemical Societies.

Phylogenetic and bioinformatic analysis of gap junction-related proteins, innexins, pannexins and connexins.

PubMed

Fushiki, Daisuke; Hamada, Yasuo; Yoshimura, Ryoichi; Endo, Yasuhisa

2010-04-01

All multi-cellular animals, including hydra, insects and vertebrates, develop gap junctions, which communicate directly with neighboring cells. Gap junctions consist of protein families called connexins in vertebrates and innexins in invertebrates. Connexins and innexins have no homology in their amino acid sequence, but both are thought to have some similar characteristics, such as a tetra-membrane-spanning structure, formation of a channel by hexamer, and transmission of small molecules (e.g. ions) to neighboring cells. Pannexins were recently identified as a homolog of innexins in vertebrate genomes. Although pannexins are thought to share the function of intercellular communication with connexins and innexins, there is little information about the relationship among these three protein families of gap junctions. We phylgenetically and bioinformatically examined these protein families and other tetra-membrane-spanning proteins using a database and three analytical softwares. The clades formed by pannexin families do not belong to the species classification but do to paralogs of each member of pannexins. Amino acid sequences of pannexins are closely related to those of innexins but less to those of connexins. These data suggest that innexins and pannexins have a common origin, but the relationship between innexins/pannexins and connexins is as slight as that of other tetra-membrane-spanning members.

Cyclic Mechanical Loading Is Essential for Rac1-Mediated Elongation and Remodeling of the Embryonic Mitral Valve.

PubMed

Gould, Russell A; Yalcin, Huseyin C; MacKay, Joanna L; Sauls, Kimberly; Norris, Russell; Kumar, Sanjay; Butcher, Jonathan T

2016-01-11

During valvulogenesis, globular endocardial cushions elongate and remodel into highly organized thin fibrous leaflets. Proper regulation of this dynamic process is essential to maintain unidirectional blood flow as the embryonic heart matures. In this study, we tested how mechanosensitive small GTPases, RhoA and Rac1, coordinate atrioventricular valve (AV) differentiation and morphogenesis. RhoA activity and its regulated GTPase-activating protein FilGAP are elevated during early cushion formation but decreased considerably during valve remodeling. In contrast, Rac1 activity was nearly absent in the early cushions but increased substantially as the valve matured. Using gain- and loss-of-function assays, we determined that the RhoA pathway was essential for the contractile myofibroblastic phenotype present in early cushion formation but was surprisingly insufficient to drive matrix compaction during valve maturation. The Rac1 pathway was necessary to induce matrix compaction in vitro through increased cell adhesion, elongation, and stress fiber alignment. Facilitating this process, we found that acute cyclic stretch was a potent activator of RhoA and subsequently downregulated Rac1 activity via FilGAP. On the other hand, chronic cyclic stretch reduced active RhoA and downstream FilGAP, which enabled Rac1 activation. Finally, we used partial atrial ligation experiments to confirm in vivo that altered cyclic mechanical loading augmented or restricted cushion elongation and thinning, directly through potentiation of active Rac1 and active RhoA, respectively. Together, these results demonstrate that cyclic mechanical signaling coordinates the RhoA to Rac1 signaling transition essential for proper embryonic mitral valve remodeling. Copyright © 2016 Elsevier Ltd. All rights reserved.

Next Generation Protein Interactomes for Plant Systems Biology and Biomass Feedstock Research

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ecker, Joseph Robert; Trigg, Shelly; Garza, Renee

Biofuel crop cultivation is a necessary step in heading towards a sustainable future, making their genomic studies a priority. While technology platforms that currently exist for studying non-model crop species, like switch-grass or sorghum, have yielded large quantities of genomic and expression data, still a large gap exists between molecular mechanism and phenotype. The aspect of molecular activity at the level of protein-protein interactions has recently begun to bridge this gap, providing a more global perspective. Interactome analysis has defined more specific functional roles of proteins based on their interaction partners, neighborhoods, and other network features, making it possible tomore » distinguish unique modules of immune response to different plant pathogens(Jiang, Dong, and Zhang 2016). As we work towards cultivating heartier biofuel crops, interactome data will lead to uncovering crop-specific defense and development networks. However, the collection of protein interaction data has been limited to expensive, time-consuming, hard-to-scale assays that mostly require cloned ORF collections. For these reasons, we have successfully developed a highly scalable, economical, and sensitive yeast two-hybrid assay, ProCREate, that can be universally applied to generate proteome-wide primary interactome data. ProCREate enables en masse pooling and massively paralleled sequencing for the identification of interacting proteins by exploiting Cre-lox recombination. ProCREate can be used to screen ORF/cDNA libraries from feedstock plant tissues. The interactome data generated will yield deeper insight into many molecular processes and pathways that can be used to guide improvement of feedstock productivity and sustainability.« less

Cysteine-containing peptides having antioxidant properties

DOEpatents

Bielicki, John K [Castro Valley, CA

2007-05-15

The term "homology" or "homologous" means an amino acid similarity measured by the program, BLAST (Altschul et al (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:33 89 3402), and expressed as --(% identity n/n). In measuring homology between a peptide and a protein of greater size, homology is measured only in the corresponding region; that is, the protein is regarded as only having the same general length as the peptide, allowing for gaps and insertions.

Assessment of the potential pathogenicity of missense mutations identified in the GTPase-activating protein (GAP)-related domain of the neurofibromatosis type-1 (NF1) gene.

PubMed

Thomas, Laura; Richards, Mark; Mort, Matthew; Dunlop, Elaine; Cooper, David N; Upadhyaya, Meena

2012-12-01

Neurofibromatosis type-1 (NF1) is caused by constitutional mutations of the NF1 tumor-suppressor gene. Although ∼85% of inherited NF1 microlesions constitute truncating mutations, the remaining ∼15% are missense mutations whose pathological relevance is often unclear. The GTPase-activating protein-related domain (GRD) of the NF1-encoded protein, neurofibromin, serves to define its major function as a negative regulator of the Ras-MAPK (mitogen-activated protein kinase) signaling pathway. We have established a functional assay to assess the potential pathogenicity of 15 constitutional nonsynonymous NF1 missense mutations (11 novel and 4 previously reported but not functionally characterized) identified in the NF1-GRD (p.R1204G, p.R1204W, p.R1276Q, p.L1301R, p.I1307V, p.T1324N, p.E1327G, p.Q1336R, p.E1356G, p.R1391G, p.V1398D, p.K1409E, p.P1412R, p.K1436Q, p.S1463F). Individual mutations were introduced into an NF1-GRD expression vector and activated Ras was assayed by an enzyme-linked immunosorbent assay (ELISA). Ten NF1-GRD variants were deemed to be potentially pathogenic by virtue of significantly elevated levels of activated GTP-bound Ras in comparison to wild-type NF1 protein. The remaining five NF1-GRD variants were deemed less likely to be of pathological significance as they exhibited similar levels of activated Ras to the wild-type protein. These conclusions received broad support from both bioinformatic analysis and molecular modeling and serve to improve our understanding of NF1-GRD structure and function. © 2012 Wiley Periodicals, Inc.

AIP1 recruits phosphatase PP2A to ASK1 in tumor necrosis factor-induced ASK1-JNK activation.

PubMed

Min, Wang; Lin, Yan; Tang, Shibo; Yu, Luyang; Zhang, Haifeng; Wan, Ting; Luhn, Tricia; Fu, Haian; Chen, Hong

2008-04-11

Previously we have shown that AIP1 (apoptosis signal-regulating kinase [ASK]1-interacting protein 1), a novel member of the Ras-GAP protein family, facilitates dephosphorylation of ASK1 at pSer967 and subsequently 14-3-3 release from ASK1, leading to enhanced ASK1-JNK signaling. However, the phosphatase(s) responsible for ASK1 dephosphorylation at pSer967 has not been identified. In the present study, we identified protein phosphatase (PP)2A as a potential phosphatase in vascular endothelial cells (ECs). Tumor necrosis factor (TNF)-induced dephosphorylation of ASK1 pSer967 in ECs was blocked by PP2A inhibitor okadaic acid. Overexpression of PP2A catalytic subunit induced dephosphorylation of ASK1 pSer967 and activation of c-Jun N-terminal kinase (JNK). In contrast, a catalytic inactive form of PP2A or PP2A small interfering RNA blunted TNF-induced dephosphorylation of ASK1 pSer967 and activation of JNK without effects on NF-kappaB activation. Whereas AIP1, via its C2 domain, binds to ASK1, PP2A binds to the GAP domain of AIP1. Endogenous AIP1-PP2A complex can be detected in the resting state, and TNF induces a complex formation of AIP1-PP2A with ASK1. Furthermore, TNF-induced association of PP2A with ASK1 was diminished in AIP1-knockdown ECs, suggesting a critical role of AIP1 in recruiting PP2A to ASK1. TNF-signaling molecules TRAF2 and RIP1, known to be in complex with AIP1 and activate AIP1 by phosphorylating AIP1 at Ser604, are critical for TNF-induced ASK1 dephosphorylation. Finally, PP2A and AIP1 cooperatively induce activation of ASK1-JNK signaling and EC apoptosis, as demonstrated by both overexpression and small interfering RNA knockdown approaches. Taken together, our data support a critical role of PP2A-AIP1 complex in TNF-induced activation of ASK1-JNK apoptotic signaling.

Akt substrate TBC1D1 regulates GLUT1 expression through the mTOR pathway in 3T3-L1 adipocytes

PubMed Central

Zhou, Qiong L.; Jiang, Zhen Y.; Holik, John; Chawla, Anil; Hagan, G. Nana; Leszyk, John; Czech, Michael P.

2010-01-01

Multiple studies have suggested that the protein kinase Akt/PKB (protein kinase B) is required for insulin-stimulated glucose transport in skeletal muscle and adipose cells. In an attempt to understand links between Akt activation and glucose transport regulation, we applied mass spectrometry-based proteomics and bioinformatics approaches to identify potential Akt substrates containing the phospho-Akt substrate motif RXRXXpS/T. The present study describes the identification of the Rab GAP (GTPase-activating protein)-domain containing protein TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1], which is closely related to TBC1D4 [TBC domain family, member 4, also denoted AS160 (Akt substrate of 160 kDa)], as an Akt substrate that is phosphorylated at Thr590. RNAi (RNA interference)-me-diated silencing of TBC1D1 elevated basal deoxyglucose uptake by approx. 61% in 3T3-L1 mouse embryo adipocytes, while the suppression of TBC1D4 and RapGAP220 under the same conditions had little effect on basal and insulin-stimulated deoxy-glucose uptake. Silencing of TBC1D1 strongly increased expression of the GLUT1 glucose transporter but not GLUT4 in cultured adipocytes, whereas the decrease in TBC1D4 had no effect. Remarkably, loss of TBC1D1 in 3T3-L1 adipocytes activated the mTOR (mammalian target of rapamycin)-p70 S6 protein kinase pathway, and the increase in GLUT1 expression in the cells treated with TBC1D1 siRNA (small interfering RNA) was blocked by the mTOR inhibitor rapamycin. Furthermore, overexpression of the mutant TBC1D1-T590A, lacking the putative Akt/PKB phosphorylation site, inhibited insulin stimulation of p70 S6 kinase phosphorylation at Thr389, a phosphorylation induced by mTOR. Taken together, our data suggest that TBC1D1 may be involved in controlling GLUT1 glucose transporter expression through the mTOR-p70 S6 kinase pathway. PMID:18215134

Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells

PubMed Central

Cheng, Catherine; Nowak, Roberta B.; Gao, Junyuan; Sun, Xiurong; Biswas, Sondip K.; Lo, Woo-Kuen; Mathias, Richard T.

2015-01-01

The eye lens consists of layers of tightly packed fiber cells, forming a transparent and avascular organ that is important for focusing light onto the retina. A microcirculation system, facilitated by a network of gap junction channels composed of connexins 46 and 50 (Cx46 and Cx50), is hypothesized to maintain and nourish lens fiber cells. We measured lens impedance in mice lacking tropomodulin 1 (Tmod1, an actin pointed-end capping protein), CP49 (a lens-specific intermediate filament protein), or both Tmod1 and CP49. We were surprised to find that simultaneous loss of Tmod1 and CP49, which disrupts cytoskeletal networks in lens fiber cells, results in increased gap junction coupling resistance, hydrostatic pressure, and sodium concentration. Protein levels of Cx46 and Cx50 in Tmod1−/−;CP49−/− double-knockout (DKO) lenses were unchanged, and electron microscopy revealed normal gap junctions. However, immunostaining and quantitative analysis of three-dimensional confocal images showed that Cx46 gap junction plaques are smaller and more dispersed in DKO differentiating fiber cells. The localization and sizes of Cx50 gap junction plaques in DKO fibers were unaffected, suggesting that Cx46 and Cx50 form homomeric channels. We also demonstrate that gap junction plaques rest in lacunae of the membrane-associated actin-spectrin network, suggesting that disruption of the actin-spectrin network in DKO fibers may interfere with gap junction plaque accretion into micrometer-sized domains or alter the stability of large plaques. This is the first work to reveal that normal gap junction plaque localization and size are associated with normal lens coupling conductance. PMID:25740157

Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells.

PubMed

Cheng, Catherine; Nowak, Roberta B; Gao, Junyuan; Sun, Xiurong; Biswas, Sondip K; Lo, Woo-Kuen; Mathias, Richard T; Fowler, Velia M

2015-05-15

The eye lens consists of layers of tightly packed fiber cells, forming a transparent and avascular organ that is important for focusing light onto the retina. A microcirculation system, facilitated by a network of gap junction channels composed of connexins 46 and 50 (Cx46 and Cx50), is hypothesized to maintain and nourish lens fiber cells. We measured lens impedance in mice lacking tropomodulin 1 (Tmod1, an actin pointed-end capping protein), CP49 (a lens-specific intermediate filament protein), or both Tmod1 and CP49. We were surprised to find that simultaneous loss of Tmod1 and CP49, which disrupts cytoskeletal networks in lens fiber cells, results in increased gap junction coupling resistance, hydrostatic pressure, and sodium concentration. Protein levels of Cx46 and Cx50 in Tmod1(-/-);CP49(-/-) double-knockout (DKO) lenses were unchanged, and electron microscopy revealed normal gap junctions. However, immunostaining and quantitative analysis of three-dimensional confocal images showed that Cx46 gap junction plaques are smaller and more dispersed in DKO differentiating fiber cells. The localization and sizes of Cx50 gap junction plaques in DKO fibers were unaffected, suggesting that Cx46 and Cx50 form homomeric channels. We also demonstrate that gap junction plaques rest in lacunae of the membrane-associated actin-spectrin network, suggesting that disruption of the actin-spectrin network in DKO fibers may interfere with gap junction plaque accretion into micrometer-sized domains or alter the stability of large plaques. This is the first work to reveal that normal gap junction plaque localization and size are associated with normal lens coupling conductance. Copyright © 2015 the American Physiological Society.

Low Level Pro-inflammatory Cytokines Decrease Connexin36 Gap Junction Coupling in Mouse and Human Islets through Nitric Oxide-mediated Protein Kinase Cδ.

PubMed

Farnsworth, Nikki L; Walter, Rachelle L; Hemmati, Alireza; Westacott, Matthew J; Benninger, Richard K P

2016-02-12

Pro-inflammatory cytokines contribute to the decline in islet function during the development of diabetes. Cytokines can disrupt insulin secretion and calcium dynamics; however, the mechanisms underlying this are poorly understood. Connexin36 gap junctions coordinate glucose-induced calcium oscillations and pulsatile insulin secretion across the islet. Loss of gap junction coupling disrupts these dynamics, similar to that observed during the development of diabetes. This study investigates the mechanisms by which pro-inflammatory cytokines mediate gap junction coupling. Specifically, as cytokine-induced NO can activate PKCδ, we aimed to understand the role of PKCδ in modulating cytokine-induced changes in gap junction coupling. Isolated mouse and human islets were treated with varying levels of a cytokine mixture containing TNF-α, IL-1β, and IFN-γ. Islet dysfunction was measured by insulin secretion, calcium dynamics, and gap junction coupling. Modulators of PKCδ and NO were applied to determine their respective roles in modulating gap junction coupling. High levels of cytokines caused cell death and decreased insulin secretion. Low levels of cytokine treatment disrupted calcium dynamics and decreased gap junction coupling, in the absence of disruptions to insulin secretion. Decreases in gap junction coupling were dependent on NO-regulated PKCδ, and altered membrane organization of connexin36. This study defines several mechanisms underlying the disruption to gap junction coupling under conditions associated with the development of diabetes. These mechanisms will allow for greater understanding of islet dysfunction and suggest ways to ameliorate this dysfunction during the development of diabetes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

TBC-8, a Putative RAB-2 GAP, Regulates Dense Core Vesicle Maturation in Caenorhabditis elegans

PubMed Central

Hannemann, Mandy; Sasidharan, Nikhil; Hegermann, Jan; Kutscher, Lena M.; Koenig, Sabine; Eimer, Stefan

2012-01-01

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2–specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation. PMID:22654674

Requirement for XLF/Cernunnos in alignment-based gap filling by DNA polymerases lambda and mu for nonhomologous end joining in human whole-cell extracts.

PubMed

Akopiants, Konstantin; Zhou, Rui-Zhe; Mohapatra, Susovan; Valerie, Kristoffer; Lees-Miller, Susan P; Lee, Kyung-Jong; Chen, David J; Revy, Patrick; de Villartay, Jean-Pierre; Povirk, Lawrence F

2009-07-01

XLF/Cernunnos is a core protein of the nonhomologous end-joining pathway of DNA double-strand break repair. To better define the role of Cernunnos in end joining, whole-cell extracts were prepared from Cernunnos-deficient human cells. These extracts effected little joining of DNA ends with cohesive 5' or 3' overhangs, and no joining at all of partially complementary 3' overhangs that required gap filling prior to ligation. Assays in which gap-filled but unligated intermediates were trapped using dideoxynucleotides revealed that there was no gap filling on aligned DSB ends in the Cernunnos-deficient extracts. Recombinant Cernunnos protein restored gap filling and end joining of partially complementary overhangs, and stimulated joining of cohesive ends more than twentyfold. XLF-dependent gap filling was nearly eliminated by immunodepletion of DNA polymerase lambda, but was restored by addition of either polymerase lambda or polymerase mu. Thus, Cernunnos is essential for gap filling by either polymerase during nonhomologous end joining, suggesting that it plays a major role in aligning the two DNA ends in the repair complex.

In silico identification of genetically attenuated vaccine candidate genes for Plasmodium liver stage.

PubMed

Kumar, Hirdesh; Frischknecht, Friedrich; Mair, Gunnar R; Gomes, James

2015-12-01

Genetically attenuated parasites (GAPs) that lack genes essential for the liver stage of the malaria parasite, and therefore cause developmental arrest, have been developed as live vaccines in rodent malaria models and recently been tested in humans. The genes targeted for deletion were often identified by trial and error. Here we present a systematic gene - protein and transcript - expression analyses of several Plasmodium species with the aim to identify candidate genes for the generation of novel GAPs. With a lack of liver stage expression data for human malaria parasites, we used data available for liver stage development of Plasmodium yoelii, a rodent malaria model, to identify proteins expressed in the liver stage but absent from blood stage parasites. An orthology-based search was then employed to identify orthologous proteins in the human malaria parasite Plasmodium falciparum resulting in a total of 310 genes expressed in the liver stage but lacking evidence of protein expression in blood stage parasites. Among these 310 possible GAP candidates, we further studied Plasmodium liver stage proteins by phyletic distribution and functional domain analyses and shortlisted twenty GAP-candidates; these are: fabB/F, fabI, arp, 3 genes encoding subunits of the PDH complex, dnaJ, urm1, rS5, ancp, mcp, arh, gk, lisp2, valS, palm, and four conserved Plasmodium proteins of unknown function. Parasites lacking one or several of these genes might yield new attenuated malaria parasites for experimental vaccination studies. Copyright © 2015 Elsevier B.V. All rights reserved.

Disruption of hippocampus-regulated behavioural and cognitive processes by heterozygous constitutive deletion of SynGAP.

PubMed

Muhia, Mary; Yee, Benjamin K; Feldon, Joram; Markopoulos, Foivos; Knuesel, Irene

2010-02-01

The brain-specific Ras/Rap-GTPase activating protein (SynGAP) is a prime candidate linking N-methyl-d-aspartate receptors to the regulation of the ERK/MAP kinase signalling cascade, suggested to be essential for experience-dependent synaptic plasticity. Here, we evaluated the behavioural phenotype of SynGAP heterozygous knockout mice (SG(+/-)), expressing roughly half the normal levels of SynGAP. In the cognitive domain, SG(+/-) mice demonstrated severe working and reference memory deficits in the radial arm maze task, a mild impairment early in the transfer test of the water maze task, and a deficiency in spontaneous alternation in an elevated T-maze. In the non-cognitive domain, SG(+/-) mice were hyperactive in the open field and appeared less anxious in the elevated plus maze test. In contrast, object recognition memory performance was not impaired in SG(+/-) mice. The reduction in SynGAP thus resulted in multiple behavioural traits suggestive of aberrant cognitive and non-cognitive processes normally mediated by the hippocampus. Immunohistochemical evaluation further revealed a significant reduction in calbindin-positive interneurons in the hippocampus and doublecortin-positive neurons in the dentate gyrus of adult SG(+/-) mice. Heterozygous constitutive deletion of SynGAP is therefore associated with notable behavioural as well as morphological phenotypes indicative of hippocampal dysfunction. Any suggestion of a possible causal link between them however remains a matter for further investigation.

Inference of RhoGAP/GTPase regulation using single-cell morphological data from a combinatorial RNAi screen.

PubMed

Nir, Oaz; Bakal, Chris; Perrimon, Norbert; Berger, Bonnie

2010-03-01

Biological networks are highly complex systems, consisting largely of enzymes that act as molecular switches to activate/inhibit downstream targets via post-translational modification. Computational techniques have been developed to perform signaling network inference using some high-throughput data sources, such as those generated from transcriptional and proteomic studies, but comparable methods have not been developed to use high-content morphological data, which are emerging principally from large-scale RNAi screens, to these ends. Here, we describe a systematic computational framework based on a classification model for identifying genetic interactions using high-dimensional single-cell morphological data from genetic screens, apply it to RhoGAP/GTPase regulation in Drosophila, and evaluate its efficacy. Augmented by knowledge of the basic structure of RhoGAP/GTPase signaling, namely, that GAPs act directly upstream of GTPases, we apply our framework for identifying genetic interactions to predict signaling relationships between these proteins. We find that our method makes mediocre predictions using only RhoGAP single-knockdown morphological data, yet achieves vastly improved accuracy by including original data from a double-knockdown RhoGAP genetic screen, which likely reflects the redundant network structure of RhoGAP/GTPase signaling. We consider other possible methods for inference and show that our primary model outperforms the alternatives. This work demonstrates the fundamental fact that high-throughput morphological data can be used in a systematic, successful fashion to identify genetic interactions and, using additional elementary knowledge of network structure, to infer signaling relations.

Polycyclic aromatic hydrocarbon-induced signaling events relevant to inflammation and tumorigenesis in lung cells are dependent on molecular structure.

PubMed

Osgood, Ross S; Upham, Brad L; Hill, Thomas; Helms, Katherine L; Velmurugan, Kalpana; Babica, Pavel; Bauer, Alison K

2014-01-01

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental and occupational toxicants, which are a major human health concern in the U.S. and abroad. Previous research has focused on the genotoxic events caused by high molecular weight PAHs, but not on non-genotoxic events elicited by low molecular weight PAHs. We used an isomeric pair of low molecular weight PAHs, namely 1-Methylanthracene (1-MeA) and 2-Methylanthracene (2-MeA), in which only 1-MeA possessed a bay-like region, and hypothesized that 1-MeA, but not 2-MeA, would affect non-genotoxic endpoints relevant to tumor promotion in murine C10 lung cells, a non-tumorigenic type II alveolar pneumocyte and progenitor cell type of lung adenocarcinoma. The non-genotoxic endpoints assessed were dysregulation of gap junction intercellular communication function and changes in the major pulmonary connexin protein, connexin 43, using fluorescent redistribution and immunoblots, activation of mitogen activated protein kinases (MAPK) using phosphospecific MAPK antibodies for immunoblots, and induction of inflammatory genes using quantitative RT-PCR. 2-MeA had no effect on any of the endpoints, but 1-MeA dysregulated gap junctional communication in a dose and time dependent manner, reduced connexin 43 protein expression, and altered membrane localization. 1-MeA also activated ERK1/2 and p38 MAP kinases. Inflammatory genes, such as cyclooxygenase 2, and chemokine ligand 2 (macrophage chemoattractant 2), were also upregulated in response to 1-MeA only. These results indicate a possible structure-activity relationship of these low molecular weight PAHs relevant to non-genotoxic endpoints of the promoting aspects of cancer. Therefore, our novel findings may improve the ability to predict outcomes for future studies with additional toxicants and mixtures, identify novel targets for biomarkers and chemotherapeutics, and have possible implications for future risk assessment for these PAHs.

Podophyllum hexandrum (Himalayan mayapple) extract provides radioprotection by modulating the expression of proteins associated with apoptosis.

PubMed

Kumar, Raj; Singh, Pankaj Kumar; Sharma, Ashok; Prasad, Jagdish; Sagar, Ravinder; Singh, Surender; Arora, Rajesh; Sharma, Rakesh Kumar

2005-08-01

Podophyllum hexandrum Royale (Himalayan mayapple), a high-altitude Himalayan plant, has been shown to provide over 80% whole-body radioprotection in mice. To investigate the radioprotective potential of P. hexandrum at the molecular level, expression patterns of various proteins associated with apoptosis were studied in the spleen of male Swiss albino strain A mice by immunoblotting. Treatment with P. hexandrum [200 mg/kg of body weight; an ethanolic 50% (w/v) extract delivered intraperitoneally] 2 h before irradiation resulted in MAPKAP (mitogen-activated protein kinase-activated protein) kinase-2 activation along with HSF-1 (heat-shock transcription factor-1), leading to up-regulation of HSP-70 (heat-shock protein-70) as compared with sham-irradiated (10 Gy) mice. Strong inhibition of AIF (apoptosis-inducing factor) expression was observed in the mice treated with P. hexandrum 2 h before irradiation as compared with the sham-irradiated group. Inhibition in the translocation of free NF-kappaB (nuclear factor kappaB) from cytoplasm to nucleus was observed upon P. hexandrum pretreatment 2 h before irradiation when compared with radiation-treated mice. P. hexandrum pre-treatment (2 h before irradiation) resulted in inhibition of NF-kappaB translocation, and the expression of tumour suppressor protein p53 was observed to be down-regulated as compared with sham-irradiated control. An increase in the expression of proteins responsible for cell proliferation [Bcl-2 (B-cell chronic lymphocytic lymphoma 2), Ras-GAP (Ras-GTPase-activating protein) and PCNA (proliferating cell nuclear antigen)] was observed in the P. hexandrum-pretreated irradiated mice as compared with sham-irradiated controls. Caspase 3 activation resulted PARP [poly(ADP-ribose) DNA polymerase] cleavage, and DNA degradation was strongly inhibited in the mice treated with P. hexandrm (+/-irradiation) as compared with the mice treated with radiation (+/-heat shock). The present study thus clearly demonstrated that P. hexandrum extract provides protection from gamma-radiation by the modulation of expression of proteins associated with cell death.

Use of designed sequences in protein structure recognition.

PubMed

Kumar, Gayatri; Mudgal, Richa; Srinivasan, Narayanaswamy; Sandhya, Sankaran

2018-05-09

Knowledge of the protein structure is a pre-requisite for improved understanding of molecular function. The gap in the sequence-structure space has increased in the post-genomic era. Grouping related protein sequences into families can aid in narrowing the gap. In the Pfam database, structure description is provided for part or full-length proteins of 7726 families. For the remaining 52% of the families, information on 3-D structure is not yet available. We use the computationally designed sequences that are intermediately related to two protein domain families, which are already known to share the same fold. These strategically designed sequences enable detection of distant relationships and here, we have employed them for the purpose of structure recognition of protein families of yet unknown structure. We first measured the success rate of our approach using a dataset of protein families of known fold and achieved a success rate of 88%. Next, for 1392 families of yet unknown structure, we made structural assignments for part/full length of the proteins. Fold association for 423 domains of unknown function (DUFs) are provided as a step towards functional annotation. The results indicate that knowledge-based filling of gaps in protein sequence space is a lucrative approach for structure recognition. Such sequences assist in traversal through protein sequence space and effectively function as 'linkers', where natural linkers between distant proteins are unavailable. This article was reviewed by Oliviero Carugo, Christine Orengo and Srikrishna Subramanian.

Gap junction intercellular communication mediated by connexin43 in astrocytes is essential for their resistance to oxidative stress.

PubMed

Le, Hoa T; Sin, Wun Chey; Lozinsky, Shannon; Bechberger, John; Vega, José Luis; Guo, Xu Qiu; Sáez, Juan C; Naus, Christian C

2014-01-17

Oxidative stress induced by reactive oxygen species (ROS) is associated with various neurological disorders including aging, neurodegenerative diseases, as well as traumatic and ischemic insults. Astrocytes have an important role in the anti-oxidative defense in the brain. The gap junction protein connexin43 (Cx43) forms intercellular channels as well as hemichannels in astrocytes. In the present study, we investigated the contribution of Cx43 to astrocytic death induced by the ROS hydrogen peroxide (H2O2) and the mechanism by which Cx43 exerts its effects. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased ROS-induced astrocytic death, supporting a cell protective effect of functional Cx43 channels. H2O2 transiently increased hemichannel activity, but reduced gap junction intercellular communication (GJIC). GJIC in wild-type astrocytes recovered after 7 h, but was absent in Cx43 knock-out astrocytes. Blockage of Cx43 hemichannels incompletely inhibited H2O2-induced hemichannel activity, indicating the presence of other hemichannel proteins. Panx1, which is predicted to be a major hemichannel contributor in astrocytes, did not appear to have any cell protective effect from H2O2 insults. Our data suggest that GJIC is important for Cx43-mediated ROS resistance. In contrast to hypoxia/reoxygenation, H2O2 treatment decreased the ratio of the hypophosphorylated isoform to total Cx43 level. Cx43 has been reported to promote astrocytic death induced by hypoxia/reoxygenation. We therefore speculate the increase in Cx43 dephosphorylation may account for the facilitation of astrocytic death. Our findings suggest that the role of Cx43 in response to cellular stress is dependent on the activation of signaling pathways leading to alteration of Cx43 phosphorylation states.

Regulating unfolded protein response activator HAC1p for production of thermostable raw-starch hydrolyzing α-amylase in Pichia pastoris.

PubMed

Huang, Mengmeng; Gao, Yanyun; Zhou, Xiangshan; Zhang, Yuanxing; Cai, Menghao

2017-03-01

Unfolded protein response (UPR) usually happens when expressing heterologous proteins in high level, which may help cells to facilitate protein processing. Here, we evaluated the effects of the UPR activator HAC1p on a raw-starch hydrolyzing α-amylase (Gs4j-amyA), so as to improve heterologous production of the enzyme in Pichia pastoris. The gene (amyA) encoding Gs4j-amyA was first codon-optimized and expressed in P. pastoris under the control of the AOX1 promoter. A high gene dosage (12 copies) of amyA facilitated amylase expression which produced an enzyme activity of 305 U/ml. A spliced HAC1 encoding an UPR activator HAC1p was then co-expressed and the dosage effects of HAC1 on amylase expression was investigated. Six copies of HAC1 driven by AOX1 promoter produced a high amylase activity of 2200 U/ml, further increasing by 621%. However, excessive gene dosages driven by the same promoter led to a titration effect of its transcription factors and decreased the amount of amyA transcripts. Thus, constitutive expression of HAC1 by GAP promotor was further involved and Gs4j-amyA activity reached 3700 U/ml finally, which was further increased by 68.2%. Moreover, Gs4j-amyA was glycosylated in P. pastoris which generated higher enzyme activity than that in E. coli. Generally, regulating HAC1p expression by different strategies enhanced amylase production by 11.1 folds, indicating a reference for expression of other proteins in P. pastoris.

ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling*

PubMed Central

Kanamarlapudi, Venkateswarlu; Thompson, Aiysha; Kelly, Eamonn; López Bernal, Andrés

2012-01-01

The luteinizing hormone chorionic gonadotropin receptor (LHCGR) is a Gs-coupled GPCR that is essential for the maturation and function of the ovary and testis. LHCGR is internalized following its activation, which regulates the biological responsiveness of the receptor. Previous studies indicated that ADP-ribosylation factor (ARF)6 and its GTP-exchange factor (GEF) cytohesin 2 regulate LHCGR internalization in follicular membranes. However, the mechanisms by which ARF6 and cytohesin 2 regulate LHCGR internalization remain incompletely understood. Here we investigated the role of the ARF6 signaling pathway in the internalization of heterologously expressed human LHCGR (HLHCGR) in intact cells using a combination of pharmacological inhibitors, siRNA and the expression of mutant proteins. We found that human CG (HCG)-induced HLHCGR internalization, cAMP accumulation and ARF6 activation were inhibited by Gallein (βγ inhibitor), Wortmannin (PI 3-kinase inhibitor), SecinH3 (cytohesin ARF GEF inhibitor), QS11 (an ARF GAP inhibitor), an ARF6 inhibitory peptide and ARF6 siRNA. However, Dynasore (dynamin inhibitor), the dominant negative mutants of NM23-H1 (dynamin activator) and clathrin, and PBP10 (PtdIns 4,5-P2-binding peptide) inhibited agonist-induced HLHCGR and cAMP accumulation but not ARF6 activation. These results indicate that heterotrimeric G-protein, phosphatidylinositol (PI) 3-kinase (PI3K), cytohesin ARF GEF and ARF GAP function upstream of ARF6 whereas dynamin and clathrin act downstream of ARF6 in the regulation of HCG-induced HLHCGR internalization and signaling. In conclusion, we have identified the components and molecular details of the ARF6 signaling pathway required for agonist-induced HLHCGR internalization. PMID:22523074

Transient suppression of gap junctional intercellular communication after exposure to 100-nanosecond pulsed electric fields.

PubMed

Steuer, Anna; Schmidt, Anke; Labohá, Petra; Babica, Pavel; Kolb, Juergen F

2016-12-01

Gap junctional intercellular communication (GJIC) is an important mechanism that is involved and affected in many diseases and injuries. So far, the effect of nanosecond pulsed electric fields (nsPEFs) on the communication between cells was not investigated. An in vitro approach is presented with rat liver epithelial WB-F344 cells grown and exposed in a monolayer. In order to observe sub-lethal effects, cells were exposed to pulsed electric fields with a duration of 100ns and amplitudes between 10 and 20kV/cm. GJIC strongly decreased within 15min after treatment but recovered within 24h. Gene expression of Cx43 was significantly decreased and associated with a reduced total amount of Cx43 protein. In addition, MAP kinases p38 and Erk1/2, involved in Cx43 phosphorylation, were activated and Cx43 became hyperphosphorylated. Immunofluorescent staining of Cx43 displayed the disassembly of gap junctions. Further, a reorganization of the actin cytoskeleton was observed whereas tight junction protein ZO-1 was not significantly affected. All effects were field- and time-dependent and most pronounced within 30 to 60min after treatment. A better understanding of a possible manipulation of GJIC by nsPEFs might eventually offer a possibility to develop and improve treatments. Copyright © 2016 Elsevier B.V. All rights reserved.

Connexin Communication Compartments and Wound Repair in Epithelial Tissue.

PubMed

Chanson, Marc; Watanabe, Masakatsu; O'Shaughnessy, Erin M; Zoso, Alice; Martin, Patricia E

2018-05-03

Epithelial tissues line the lumen of tracts and ducts connecting to the external environment. They are critical in forming an interface between the internal and external environment and, following assault from environmental factors and pathogens, they must rapidly repair to maintain cellular homeostasis. These tissue networks, that range from a single cell layer, such as in airway epithelium, to highly stratified and differentiated epithelial surfaces, such as the epidermis, are held together by a junctional nexus of proteins including adherens, tight and gap junctions, often forming unique and localised communication compartments activated for localised tissue repair. This review focuses on the dynamic changes that occur in connexins, the constituent proteins of the intercellular gap junction channel, during wound-healing processes and in localised inflammation, with an emphasis on the lung and skin. Current developments in targeting connexins as corrective therapies to improve wound closure and resolve localised inflammation are also discussed. Finally, we consider the emergence of the zebrafish as a concerted whole-animal model to study, visualise and track the events of wound repair and regeneration in real-time living model systems.

Basolateral Endocytic Recycling Requires RAB-10 and AMPH-1 Mediated Recruitment of RAB-5 GAP TBC-2 to Endosomes

PubMed Central

Liu, Ou; Grant, Barth D.

2015-01-01

The small GTPase RAB-5/Rab5 is a master regulator of the early endosome, required for a myriad of coordinated activities, including the degradation and recycling of internalized cargo. Here we focused on the recycling function of the early endosome and the regulation of RAB-5 by GAP protein TBC-2 in the basolateral C. elegans intestine. We demonstrate that downstream basolateral recycling regulators, GTPase RAB-10/Rab10 and BAR domain protein AMPH-1/Amphiphysin, bind to TBC-2 and help to recruit it to endosomes. In the absence of RAB-10 or AMPH-1 binding to TBC-2, RAB-5 membrane association is abnormally high and recycling cargo is trapped in early endosomes. Furthermore, the loss of TBC-2 or AMPH-1 leads to abnormally high spatial overlap of RAB-5 and RAB-10. Taken together our results indicate that RAB-10 and AMPH-1 mediated down-regulation of RAB-5 is an important step in recycling, required for cargo exit from early endosomes and regulation of early endosome–recycling endosome interactions. PMID:26393361

Opposite effects of the gap junction blocker octanol on focal cerebral ischemia occluded for different durations.

PubMed

Ding, Wenting; Zhou, Lequan; Liu, Wei; Guan, Li; Li, Xiaoying; Liu, Haimei; Yan, Fuman; Xu, Jinwen; Zeng, Weiyong; Qiu, Min

2014-06-01

Protectants and executioners have been demonstrated to be used by gap junctions in focal cerebral ischemia. Certain researchers hypothesized that the opposite role of gap junctions may be associated with the injury extent, which has been demonstrated to be highly correlated with occlusion duration. In order to examine this hypothesis directly, the effects of octanol, a frequently used drug, were examined to investigate the role of gap junctions, in rats following middle cerebral artery occlusion (MCAO) for 30 min/2 h and 24 h reperfusion, respectively. Octanol significantly reduced the infarct volume following 2 h of occlusion concomitant with lower neurological deficits, whereas it enlarged the infarct volume following 30 min of occlusion. Consistently, octanol attenuated the number of transferase dUTP nick-end labeling (TUNEL) positive neurons in the hippocampal CA1 region following 2 h of occlusion, while opposite effects were observed for 30 min of occlusion. Further immunohistochemical studies demonstrated that the expression of B-cell leukemia-2 (Bcl-2, anti-apoptotic protein) was upregulated and that Bcl-2-associated X (Bax, proapoptotic protein) was downregulated following 2 h of occlusion in the octanol group compared with the ischemic group. Conversely, octanol downregulated the expression of the Bcl-2 protein concomitant with increased Bax protein following 30 min of occlusion. These results indicated that the gap junction blocker octanol can protect against ischemic injury following long-term occlusion, however, can aggravate ischemic injury following short-term occlusion.

Molecular dynamics simulations reveal the conformational dynamics of Arabidopsis thaliana BRI1 and BAK1 receptor-like kinases.

PubMed

Moffett, Alexander S; Bender, Kyle W; Huber, Steven C; Shukla, Diwakar

2017-07-28

The structural motifs responsible for activation and regulation of eukaryotic protein kinases in animals have been studied extensively in recent years, and a coherent picture of their activation mechanisms has begun to emerge. In contrast, non-animal eukaryotic protein kinases are not as well understood from a structural perspective, representing a large knowledge gap. To this end, we investigated the conformational dynamics of two key Arabidopsis thaliana receptor-like kinases, brassinosteroid-insensitive 1 (BRI1) and BRI1-associated kinase 1 (BAK1), through extensive molecular dynamics simulations of their fully phosphorylated kinase domains. Molecular dynamics simulations calculate the motion of each atom in a protein based on classical approximations of interatomic forces, giving researchers insight into protein function at unparalleled spatial and temporal resolutions. We found that in an otherwise "active" BAK1 the αC helix is highly disordered, a hallmark of deactivation, whereas the BRI1 αC helix is moderately disordered and displays swinging behavior similar to numerous animal kinases. An analysis of all known sequences in the A. thaliana kinome found that αC helix disorder may be a common feature of plant kinases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abbasifar, Reza; Griffiths, Mansel W.; Sabour, Parviz M.

Cronobacter sakazakii is a Gram-negative pathogen found in milk-based formulae that causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage vB{sub C}saM{sub G}AP32 (GAP32), which possesses the second largest sequenced phage genome (358,663 bp). A total of 571 genes including 545 protein coding sequences and 26 tRNAs were identified, thus more genes than in the smallest bacterium, Mycoplasma genitalium G37. BLASTP and HHpred searches, together with proteomic analyses reveal that only 23.9% of the putative proteins havemore » defined functions. Some of the unique features of this phage include: a chromosome condensation protein, two copies of the large subunit terminase, a predicted signal-arrest-release lysin; and an RpoD-like protein, which is possibly involved in the switch from immediate early to delayed early transcription. Its closest relatives are all extremely large myoviruses, namely coliphage PBECO4 and Klebsiella phage vB{sub K}leM-RaK2, with whom it shares approximately 44% homologous proteins. Since the homologs are not evenly distributed, we propose that these three phages belong to a new subfamily. - Highlights: • Cronobacter sakazakii phage vB{sub C}saM{sub G}AP32 has a genome of 358,663 bp. • It encodes 545 proteins which is more than Mycoplasma genitalium G37. • It is a member of the Myoviridae. • It is peripherally related to coliphage PBECO4 and Klebsiella phage vB{sub K}leM-RaK2. • GAP32 encodes a chromosome condensation protein.« less

Effects of ageing and streptozotocin-induced diabetes on connexin43 and P2 purinoceptor expression in the rat corpora cavernosa and urinary bladder.

PubMed

Suadicani, Sylvia O; Urban-Maldonado, Marcia; Tar, Moses T; Melman, Arnold; Spray, David C

2009-06-01

To investigate whether ageing and diabetes alter the expression of the gap junction protein connexin43 (Cx43) and of particular purinoceptor (P2R) subtypes in the corpus cavernosum and urinary bladder, and determine whether changes in expression of these proteins correlate with development of erectile and bladder dysfunction in diabetic and ageing rats. Erectile and bladder function of streptozotocin (STZ)-induced diabetic, insulin-treated and age-matched control Fischer-344 rats were evaluated 2, 4 and 8 months after diabetes induction by in vivo cystometry and cavernosometry. Corporal and bladder tissue were then isolated at each of these sample times and protein expression levels of Cx43 and of various P2R subtypes were determined by Western blotting. In the corpora of control rats ageing was accompanied by a significant decrease in Cx43 and P2X(1)R, and increase in P2X(7)R expression. There was decreased Cx43 and increased P2Y(4)R expression in the ageing control rat bladder. There was a significant negative correlation between erectile capacity and P2X(1)R expression levels, and a positive correlation between bladder spontaneous activity and P2Y(4)R expression levels. There was already development of erectile dysfunction and bladder overactivity at 2 months after inducing diabetes, the earliest sample measured in the study. The development of these urogenital complications was accompanied by significant decreases in Cx43, P2Y(2)R, P2X(4)R and increase in P2X(1)R expression in the corpora, and by a doubling in Cx43 and P2Y(2)R, and significant increase in P2Y(4)R expression in the bladder. Changes in Cx43 and P2R expression were largely prevented by insulin therapy. Ageing and diabetes mellitus markedly altered the expression of the gap junction protein Cx43 and of particular P2R subtypes in the rat penile corpora and urinary bladder. These changes in Cx43 and P2R expression provide the molecular substrate for altered gap junction and purinergic signalling in these tissues, and thus probably contribute to the early development of erectile dysfunction and higher detrusor activity in ageing and in diabetic rats.

Specialized sorting of GLUT4 and its recruitment to the cell surface are independently regulated by distinct Rabs

PubMed Central

Sadacca, L. Amanda; Bruno, Joanne; Wen, Jennifer; Xiong, Wenyong; McGraw, Timothy E.

2013-01-01

Adipocyte glucose uptake in response to insulin is essential for physiological glucose homeostasis: stimulation of adipocytes with insulin results in insertion of the glucose transporter GLUT4 into the plasma membrane and subsequent glucose uptake. Here we establish that RAB10 and RAB14 are key regulators of GLUT4 trafficking that function at independent, sequential steps of GLUT4 translocation. RAB14 functions upstream of RAB10 in the sorting of GLUT4 to the specialized transport vesicles that ferry GLUT4 to the plasma membrane. RAB10 and its GTPase-activating protein (GAP) AS160 comprise the principal signaling module downstream of insulin receptor activation that regulates the accumulation of GLUT4 transport vesicles at the plasma membrane. Although both RAB10 and RAB14 are regulated by the GAP activity of AS160 in vitro, only RAB10 is under the control of AS160 in vivo. Insulin regulation of the pool of RAB10 required for GLUT4 translocation occurs through regulation of AS160, since activation of RAB10 by DENND4C, its GTP exchange factor, does not require insulin stimulation. PMID:23804653

Mechanotransductive Regulation of Gap-Junction Activity Between MLO-Y4 Osteocyte-Like and MC3T3-E1 Osteoblast-Like Cells in Three-Dimensional Co-Culture.

NASA Technical Reports Server (NTRS)

Juran, C. M.; Blaber, E. A.; Almeida, E. A. C.

2016-01-01

Cell and animal studies conducted onboard the International Space Station and formerly on Shuttle flights have provided groundbreaking data illuminating the deleterious biological response of bone to mechanical unloading. However the intercellular communicative mechanisms associated with the regulation of bone synthesis and bone resorption cells are still largely unknown. Connexin-43 (CX43), a gap junction protein, is hypothesized to play a significant role in osteoblast and osteocyte signaling. The purpose of this investigation was to evaluate within a novel three-dimensional microenvironment how the osteocyte-osteoblast gap-junction expression changes when cultures are exposed to exaggerated mechanical load. MLO-Y4 osteocyte-like cells were cultured on a 3D-Biotek polystyrene insert and placed in direct contact with an MC3T3-E1 pre-osteoblast co-cultured monolayer and exposed to 48 h of mechanical stimulation (pulsatile fluid flow (PFF) or monolayer cyclic stretch (MCS)) then evaluated for viability, proliferation, metabolism, and CX43 expression. Mono-cultured MLO-Y4 and MC3T3-E1 control experiments were conducted under PFF and MCS stimulation to observe how strain application stimuli (PFF cell membrane shear or MCS cell focal adhesion/attachment loading) initiates different signaling pathways or downstream regulatory controls. TotalLive cell count, viability and metabolic reduction (Trypan Blue, LIVEDead and Alamar Blue analysis respectively) indicate that mechanical activation of MC3T3-E1 cells inhibits proliferation while maintaining an average 1.04E4 reductioncell metabolic rate, *p0.05 n4. MLO-Y4s in monolayer culture increase in number when exposed to MCS loading but the percent of live cells within the population is low (46.3 total count, *p0.05 n4), these results may indicate an apoptotic signaling cascade. PFF stimulation of the three-dimensional co-cultures elicits a universal increase in CX43 in MLO-Y4 and MC3T3-E1 cells, illustrated by immunohistological observation. Increased CX43 expression is also observed with the three-dimensional co-cultures with MC3T3-E1 MCS stimulation but the increased gap-junction protein presence was limited to the osteoblast-osteocyte interface region. Previously reported PCR evaluation of osteogenic markers further corroborate that the co-cultured populations communicative networks play a role in translating mechanical signals to molecular messaging. These findings suggests an osteocyte-osteoblast gap-junction signaling feedback mechanism may regulate mechanotransduction of apoptosis initiation and transcription of cytokine signaling proteins responsible for stem cell niche recruitment much more directly than previously believed.

1H, 15N, 13C resonance assignment of human GAP-43.

PubMed

Flamm, Andrea Gabriele; Żerko, Szymon; Zawadzka-Kazimierczuk, Anna; Koźmiński, Wiktor; Konrat, Robert; Coudevylle, Nicolas

2016-04-01

GAP-43 is a 25 kDa neuronal intrinsically disordered protein, highly abundant in the neuronal growth cone during development and regeneration. The exact molecular function(s) of GAP-43 remains unclear but it appears to be involved in growth cone guidance and actin cytoskeleton organization. Therefore, GAP-43 seems to play an important role in neurotransmitter vesicle fusion and recycling, long-term potentiation, spatial memory formation and learning. Here we report the nearly complete assignment of recombinant human GAP-43.

The Oncogenic Role of RhoGAPs in Basal-Like Breast Cancer

DTIC Science & Technology

2015-02-01

cell lines, and mouse models . c) In vivo tumorigenesis and metastasis assays. Milestones: Identify whether ArhGAP11A and RacGAP1 can promote tumor growth...also upregulated in basal (C3(I)-Tag) but not luminal (MMTV-Neu) genetically- engineered mouse models (Fig. 1B). At the protein level, RacGAP1 was...hypothesis that these RhoGAPs are indeed playing an oncogenic role in these cells. Human Tumors Mouse Model Tumors Normal Luminal A Basal-like Normal

Biochemistry of Mitochondrial Coenzyme Q Biosynthesis.

PubMed

Stefely, Jonathan A; Pagliarini, David J

2017-10-01

Coenzyme Q (CoQ, ubiquinone) is a redox-active lipid produced across all domains of life that functions in electron transport and oxidative phosphorylation and whose deficiency causes human diseases. Yet, CoQ biosynthesis has not been fully defined in any organism. Several proteins with unclear molecular functions facilitate CoQ biosynthesis through unknown means, and multiple steps in the pathway are catalyzed by currently unidentified enzymes. Here we highlight recent progress toward filling these knowledge gaps through both traditional biochemistry and cutting-edge 'omics' approaches. To help fill the remaining gaps, we present questions framed by the recently discovered CoQ biosynthetic complex and by putative biophysical barriers. Mapping CoQ biosynthesis, metabolism, and transport pathways has great potential to enhance treatment of numerous human diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of 18beta-glycyrrhetinic acid on the junctional complex and steroidogenesis in rat adrenocortical cells.

PubMed

Huang, Shih-Horng; Wu, Jiahn-Chun; Hwang, Ra-Der; Yeo, Hui-Lin; Wang, Seu-Mei

2003-09-01

Cellular junctions play important roles in cell differentiation, signal transduction, and cell function. This study investigated their function in steroid secretion by adrenal cells. Immunofluorescence staining revealed the presence of gap junctions and adherens junctions between adrenal cells. The major gap junction protein, connexin43, was seen as a linear dotted pattern of the typical gap junction plaques, in contrast to alpha-, beta-, and gamma-catenin, which were seen as continuous, linear staining of cell-cell adherens junction. Treatment with 18beta-glycyrrhetinic acid, a gap junction inhibitor, reduced the immunoreactivity of these proteins in a time- and dose-dependent manner, and caused the gap junction and adherens junction to separate longitudinally from the cell-cell contact sites, indicating the structural interdependency of these two junctions. Interestingly, 18beta-glycyrrhetinic acid stimulated a two- to three-fold increase in steroid production in these adrenal cells lacking intact cell junctions. These data raise the question of the necessity for cell communication for the endocrine function of adrenal cells. Pharmacological analyses indicated that the steroidogenic effect of 18beta-glycyrrhetinic acid was partially mediated by extracellular signal-related kinase and calcium/calmodulin-dependent kinase, a pathway distinct from the protein kinase A signaling pathway already known to mediate steroidogenesis in adrenal cells. Copyright 2003 Wiley-Liss, Inc.

Antitumor Activity of Rat Mesenchymal Stem Cells during Direct or Indirect Co-Culturing with C6 Glioma Cells.

PubMed

Gabashvili, A N; Baklaushev, V P; Grinenko, N F; Mel'nikov, P A; Cherepanov, S A; Levinsky, A B; Chehonin, V P

2016-02-01

The tumor-suppressive effect of rat mesenchymal stem cells against low-differentiated rat C6 glioma cells during their direct and indirect co-culturing and during culturing of C6 glioma cells in the medium conditioned by mesenchymal stem cells was studied in an in vitro experiment. The most pronounced antitumor activity of mesenchymal stem cells was observed during direct co-culturing with C6 glioma cells. The number of live C6 glioma cells during indirect co-culturing and during culturing in conditioned medium was slightly higher than during direct co-culturing, but significantly differed from the control (C6 glioma cells cultured in medium conditioned by C6 glioma cells). The cytotoxic effect of medium conditioned by mesenchymal stem cells was not related to medium depletion by glioma cells during their growth. The medium conditioned by other "non-stem" cells (rat astrocytes and fibroblasts) produced no tumor-suppressive effect. Rat mesenchymal stem cells, similar to rat C6 glioma cells express connexin 43, the main astroglial gap junction protein. During co-culturing, mesenchymal stem cells and glioma C6 cells formed functionally active gap junctions. Gap junction blockade with connexon inhibitor carbenoxolone attenuated the antitumor effect observed during direct co-culturing of C6 glioma cells and mesenchymal stem cells to the level produced by conditioned medium. Cell-cell signaling mediated by gap junctions can be a mechanism of the tumor-suppressive effect of mesenchymal stem cells against C6 glioma cells. This phenomenon can be used for the development of new methods of cell therapy for high-grade malignant gliomas.

Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster.

PubMed

Rodriguez-Fernandez, Imilce A; Dell'Angelica, Esteban C

2015-01-01

The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in autophagy and small GTPase regulation.

Zoledronate promotes bone formation by blocking osteocyte-osteoblast communication during bone defect healing.

PubMed

Cui, Pingping; Liu, Hongrui; Sun, Jing; Amizuka, Norio; Sun, Qinfeng; Li, Minqi

2018-01-01

Nitrogen-containing bisphosphonates (N-BPs) are potent antiresorptive drugs and their actions on osteoclasts have been studied extensively. Recent studies have suggested that N-BPs also target bone-forming cells. However, the precise mechanism of N-BPs in osteoblasts is paradoxical, and the specific role of osteocytes is worthy of in-depth study. Here, we investigated the cellular mechanisms of N-BPs regulating bone defect healing by zoledronate (ZA). Bone histomorphometry confirmed an increase in new bone formation by systemic ZA administration. ZA induced more alkaline phosphatase-positive osteoblasts and tartrate-resistant acid phosphatase-positive osteoclasts residing on the bone surface. Inexplicably, ZA increased SOST expression in osteocytes embedded in the bone matrix, which was not compatible with the intense osteoblast activity on the bone surface. ZA induced heterogeneous osteocytes and disturbed the distribution of the osteocytic-canalicular system (OLCS). Furthermore, according to the degree of OLCS regularity, dentin matrix protein 1 reactivity had accumulated around osteocytes in the ZA group, but it was distributed evenly in the OLCS of the control group. The control group showed a dense array of the gap junction protein connexin 43. However, connexin 43 was extremely sparse after ZA administration. In summary, ZA treatment reduces gap junction connections and blocks cellular communication between osteocytes and osteoblasts. Retaining SOST expression in osteocytes leads to activation of the Wnt signaling pathway and subsequent bone formation.

Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells.

PubMed

Carbone, Annalucia; Zefferino, Roberto; Beccia, Elisa; Casavola, Valeria; Castellani, Stefano; Di Gioia, Sante; Giannone, Valentina; Seia, Manuela; Angiolillo, Antonella; Colombo, Carla; Favia, Maria; Conese, Massimo

2018-01-01

We previously found that human amniotic mesenchymal stem cells (hAMSCs) in coculture with CF immortalised airway epithelial cells (CFBE41o- line, CFBE) on Transwell® filters acquired an epithelial phenotype and led to the expression of a mature and functional CFTR protein. In order to explore the role of gap junction- (GJ-) mediated intercellular communication (GJIC) in this rescue, cocultures (hAMSC : CFBE, 1 : 5 ratio) were studied for the formation of GJIC, before and after silencing connexin 43 (Cx43), a major component of GJs. Functional GJs in cocultures were inhibited when the expression of the Cx43 protein was downregulated. Transfection of cocultures with siRNA against Cx43 resulted in the absence of specific CFTR signal on the apical membrane and reduction in the mature form of CFTR (band C), and in parallel, the CFTR-dependent chloride channel activity was significantly decreased. Cx43 downregulation determined also a decrease in transepithelial resistance and an increase in paracellular permeability as compared with control cocultures, implying that GJIC may regulate CFTR expression and function that in turn modulate airway epithelium tightness. These results indicate that GJIC is involved in the correction of CFTR chloride channel activity upon the acquisition of an epithelial phenotype by hAMSCs in coculture with CF cells.

Relating proton pumps with gap junctions: colocalization of ductin, the channel-forming subunit c of V-ATPase, with subunit a and with innexins 2 and 3 during Drosophila oogenesis.

PubMed

Lautemann, Julia; Bohrmann, Johannes

2016-07-13

Ion-transport mechanisms and gap junctions are known to cooperate in creating bioelectric phenomena, like pH gradients, voltage gradients and ion fluxes within single cells, tissues, organs, and whole organisms. Such phenomena have been shown to play regulatory roles in a variety of developmental and regenerative processes. Using Drosophila oogenesis as a model system, we aim at characterizing in detail the mechanisms underlying bioelectric phenomena in order to reveal their regulatory functions. We, therefore, investigated the stage-specific distribution patterns of V-ATPase components in relation to gap-junction proteins. We analysed the localization of the V-ATPase components ductin (subunit c) and subunit a, and the gap-junction components innexins 2 and 3, especially in polar cells, border cells, stalk cells and centripetally migrating cells. These types of follicle cells had previously been shown to exhibit characteristic patterns of membrane channels as well as membrane potential and intracellular pH. Stage-specifically, ductin and subunit a were found either colocalized or separately enriched in different regions of soma and germ-line cells. While ductin was often more prominent in plasma membranes, subunit a was more prominent in cytoplasmic and nuclear vesicles. Particularly, ductin was enriched in polar cells, stalk cells, and nurse-cell membranes, whereas subunit a was enriched in the cytoplasm of border cells, columnar follicle cells and germ-line cells. Comparably, ductin and both innexins 2 and 3 were either colocalized or separately enriched in different cellular regions. While ductin often showed a continuous membrane distribution, the distribution of both innexins was mostly punctate. Particularly, ductin was enriched in polar cells and stalk cells, whereas innexin 2 was enriched in the oolemma, and innexin 3 in centripetally migrating follicle cells. In lateral follicle-cell membranes, the three proteins were found colocalized as well as separately concentrated in presumed gap-junction plaques. Our results support the notion of a large variety of gap junctions existing in the Drosophila ovary. Moreover, since ductin is the channel-forming part of a proton pump and, like the innexins, is able to form junctional as well as non-junctional membrane channels, a plethora of cellular functions could be realized by using these proteins. The distribution and activity patterns of such membrane channels are expected to contribute to developmentally important bioelectric signals.

Molecular evolutionary analysis of vertebrate transducins: a role for amino acid variation in photoreceptor deactivation.

PubMed

Lin, Yi G; Weadick, Cameron J; Santini, Francesco; Chang, Belinda S W

2013-12-01

Transducin is a heterotrimeric G protein that plays a critical role in phototransduction in the rod and cone photoreceptor cells of the vertebrate retina. Rods, highly sensitive cells that recover from photoactivation slowly, underlie dim-light vision, whereas cones are less sensitive, recover more quickly, and underlie bright-light vision. Transducin deactivation is a critical step in photoreceptor recovery and may underlie the functional distinction between rods and cones. Rods and cones possess distinct transducin α subunits, yet they share a common deactivation mechanism, the GTPase activating protein (GAP) complex. Here, we used codon models to examine patterns of sequence evolution in rod (GNAT1) and cone (GNAT2) α subunits. Our results indicate that purifying selection is the dominant force shaping GNAT1 and GNAT2 evolution, but that GNAT2 has additionally been subject to positive selection operating at multiple phylogenetic scales; phylogeny-wide analysis identified several sites in the GNAT2 helical domain as having substantially elevated dN/dS estimates, and branch-site analysis identified several nearby sites as targets of strong positive selection during early vertebrate history. Examination of aligned GNAT and GAP complex crystal structures revealed steric clashes between several positively selected sites and the deactivating GAP complex. This suggests that GNAT2 sequence variation could play an important role in adaptive evolution of the vertebrate visual system via effects on photoreceptor deactivation kinetics and provides an alternative perspective to previous work that focused instead on the effect of GAP complex concentration. Our findings thus further the understanding of the molecular biology, physiology, and evolution of vertebrate visual systems.

Deficiency of the Survival of Motor Neuron Protein Impairs mRNA Localization and Local Translation in the Growth Cone of Motor Neurons.

PubMed

Fallini, Claudia; Donlin-Asp, Paul G; Rouanet, Jeremy P; Bassell, Gary J; Rossoll, Wilfried

2016-03-30

Spinal muscular atrophy (SMA) is a neurodegenerative disease primarily affecting spinal motor neurons. It is caused by reduced levels of the survival of motor neuron (SMN) protein, which plays an essential role in the biogenesis of spliceosomal small nuclear ribonucleoproteins in all tissues. The etiology of the specific defects in the motor circuitry in SMA is still unclear, but SMN has also been implicated in mediating the axonal localization of mRNA-protein complexes, which may contribute to the axonal degeneration observed in SMA. Here, we report that SMN deficiency severely disrupts local protein synthesis within neuronal growth cones. We also identify the cytoskeleton-associated growth-associated protein 43 (GAP43) mRNA as a new target of SMN and show that motor neurons from SMA mouse models have reduced levels ofGAP43mRNA and protein in axons and growth cones. Importantly, overexpression of two mRNA-binding proteins, HuD and IMP1, restoresGAP43mRNA and protein levels in growth cones and rescues axon outgrowth defects in SMA neurons. These findings demonstrate that SMN plays an important role in the localization and local translation of mRNAs with important axonal functions and suggest that disruption of this function may contribute to the axonal defects observed in SMA. The motor neuron disease spinal muscular atrophy (SMA) is caused by reduced levels of the survival of motor neuron (SMN) protein, which plays a key role in assembling RNA/protein complexes that are essential for mRNA splicing. It remains unclear whether defects in this well characterized housekeeping function cause the specific degeneration of spinal motor neurons observed in SMA. Here, we describe an additional role of SMN in regulating the axonal localization and local translation of the mRNA encoding growth-associated protein 43 (GAP43). This study supports a model whereby SMN deficiency impedes transport and local translation of mRNAs important for neurite outgrowth and stabilization, thus contributing to axon degeneration, muscle denervation, and motor neuron cell death in SMA. Copyright © 2016 the authors 0270-6474/16/363811-10$15.00/0.

Application of a multi-channel microfluidic chip on the simultaneous detection of DNAs by using microbead-quantum dots.

PubMed

Le, Ngoc Tam; Kim, Jong Sung

2014-12-01

Several researches have shown that cancer is caused by genetic mutations especially in genes involved in cell growth and regulation. Ras family members are frequently found in their mutated, oncogenic forms in human tumors. Mutant RAS proteins are constitutively active, owing to reduce intrinsic GTPase activity and insensitivity to GTPase-activating protein (GAPs). In total, activating mutations in the RAS genes occur in approximately 20% of all human cancers, mainly in codon 12, 13 or 61. Activating mutations in the NRAS gene not only result in the reduction of intrinsic GTPase activity but also in the induction of resistance against molecules inducing such activity. In this paper, we reported a rapid, simple and portable method for detecting the mutant types of NRAS genes codon 12 and 61 simultaneously by using bead-quantum dots (QDs) based multi-channel microfluidic chip. Probe DNAs are conjugated to bead-QDs and packed in the pillars of channels in the microfluidic chip. After injection of target DNAs and intercalating dyes, the fluorescence quenching of QDs by intercalating dye was observed due to FRET phenomena. The platform can be effortlessly applied in other biological and clinical areas.

Analyzing phorbol ester effects on gap junctional communication: a dramatic inhibition of assembly

PubMed Central

1994-01-01

The effect of 12-O-tetradeconylphorbol-13-acetate (TPA) on gap junction assembly between Novikoff hepatoma cells was examined. Cells were dissociated with EDTA to single cells and then reaggregated to form new junctions. When TPA (25 nM) was added to the cells at the onset of the 60-min reaggregation, dye transfer was detected at only 0.6% of the cell-cell interfaces compared to 72% for the untreated control and 74% for 4-alpha TPA, an inactive isomer of TPA. Freeze-fracture electron microscopy of reaggregated control cells showed interfaces containing an average of more than 600 aggregated intramembranous gap junction particles, while TPA-treated cells had no gap junctions. However, Lucifer yellow dye transfer between nondissociated cells via gap junctions was unaffected by 60 min of TPA treatment. Therefore, TPA dramatically inhibited gap junction assembly but did not alter channel gating nor enhance disassembly of preexisting gap junction structures. Short term TPA treatment (< 30 min) increased phosphorylation of the gap junction protein molecular weight of 43,000 (Cx43), but did not change the cellular level of Cx43. Cell surface biotinylation experiments suggested that TPA did not substantially reduce the plasma membrane concentration of Cx43. Therefore, the simple presence of Cx43 in the plasma membrane is not sufficient for gap junction assembly, and protein kinase C probably exerts an effect on assembly of gap junctions at the plasma membrane level. PMID:7806568

The JAK-STAT signaling pathway: input and output integration.

PubMed

Murray, Peter J

2007-03-01

Universal and essential to cytokine receptor signaling, the JAK-STAT pathway is one of the best understood signal transduction cascades. Almost 40 cytokine receptors signal through combinations of four JAK and seven STAT family members, suggesting commonality across the JAK-STAT signaling system. Despite intense study, there remain substantial gaps in understanding how the cascades are activated and regulated. Using the examples of the IL-6 and IL-10 receptors, I will discuss how diverse outcomes in gene expression result from regulatory events that effect the JAK1-STAT3 pathway, common to both receptors. I also consider receptor preferences by different STATs and interpretive problems in the use of STAT-deficient cells and mice. Finally, I consider how the suppressor of cytokine signaling (SOCS) proteins regulate the quality and quantity of STAT signals from cytokine receptors. New data suggests that SOCS proteins introduce additional diversity into the JAK-STAT pathway by adjusting the output of activated STATs that alters downstream gene activation.

Adipocytes in both brown and white adipose tissue of adult mice are functionally connected via gap junctions: implications for Chagas disease.

PubMed

Burke, Shoshana; Nagajyothi, Fnu; Thi, Mia M; Hanani, Menachem; Scherer, Philipp E; Tanowitz, Herbert B; Spray, David C

2014-11-01

Adipose tissue serves as a host reservoir for the protozoan Trypanosoma cruzi, the causative organism in Chagas disease. Gap junctions interconnect cells of most tissues, serving to synchronize cell activities including secretion in glandular tissue, and we have previously demonstrated that gap junctions are altered in various tissues and cells infected with T. cruzi. Herein, we examined the gap junction protein connexin 43 (Cx43) expression in infected adipose tissues. Adipose tissue is the largest endocrine organ of the body and is also involved in other physiological functions. In mammals, it is primarily composed of white adipocytes. Although gap junctions are a prominent feature of brown adipocytes, they have not been explored extensively in white adipocytes, especially in the setting of infection. Thus, we examined functional coupling in both white and brown adipocytes in mice. Injection of electrical current or the dye Lucifer Yellow into adipocytes within fat tissue spread to adjacent cells, which was reduced by treatment with agents known to block gap junctions. Moreover, Cx43 was detected in both brown and white fat tissue. At thirty and ninety days post-infection, Cx43 was downregulated in brown adipocytes and upregulated in white adipocytes. Gap junction-mediated intercellular communication likely contributes to hormone secretion and other functions in white adipose tissue and to nonshivering thermogenesis in brown fat, and modulation of the coupling by T. cruzi infection is expected to impact these functions. Copyright © 2014. Published by Elsevier Masson SAS.

Rasfonin, a novel 2-pyrone derivative, induces ras-mutated Panc-1 pancreatic tumor cell death in nude mice

PubMed Central

Xiao, Z; Li, L; Li, Y; Zhou, W; Cheng, J; Liu, F; Zheng, P; Zhang, Y; Che, Y

2014-01-01

Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR–Ras–Raf–MEK–ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [3H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC50=5.5 μM) than BxPC-3 cells (IC50=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras–MAPK activity could be important in its anticancer activity. PMID:24853419

Connexins: Synthesis, Post-Translational Modifications, and Trafficking in Health and Disease

PubMed Central

Vidal-Brime, Laia; Lynn, K. Sabrina

2018-01-01

Connexins are tetraspan transmembrane proteins that form gap junctions and facilitate direct intercellular communication, a critical feature for the development, function, and homeostasis of tissues and organs. In addition, a growing number of gap junction-independent functions are being ascribed to these proteins. The connexin gene family is under extensive regulation at the transcriptional and post-transcriptional level, and undergoes numerous modifications at the protein level, including phosphorylation, which ultimately affects their trafficking, stability, and function. Here, we summarize these key regulatory events, with emphasis on how these affect connexin multifunctionality in health and disease. PMID:29701678

Human immunodeficiency virus type 1 envelope proteins traffic toward virion assembly sites via a TBC1D20/Rab1-regulated pathway

PubMed Central

2012-01-01

Background The cellular activity of many factors and pathways is required to execute the complex replication cycle of the human immunodeficiency virus type 1 (HIV-1). To reveal these cellular components, several extensive RNAi screens have been performed, listing numerous 'HIV-dependency factors'. However, only a small overlap between these lists exists, calling for further evaluation of the relevance of specific factors to HIV-1 replication and for the identification of additional cellular candidates. TBC1D20, the GTPase-activating protein (GAP) of Rab1, regulates endoplasmic reticulum (ER) to Golgi trafficking, was not identified in any of these screens, and its involvement in HIV-1 replication cycle is tested here. Findings Excessive TBC1D20 activity perturbs the early trafficking of HIV-1 envelope protein through the secretory pathway. Overexpression of TBC1D20 hampered envelope processing and reduced its association with detergent-resistant membranes, entailing a reduction in infectivity of HIV-1 virion like particles (VLPs). Conclusions These findings add TBC1D20 to the network of host factors regulating HIV replication cycle. PMID:22260459

RhoGTPase signalling at epithelial tight junctions: Bridging the GAP between polarity and cancer.

PubMed

Zihni, Ceniz; Terry, Stephen James

2015-07-01

The establishment and maintenance of epithelial polarity must be correctly controlled for normal development and homeostasis. Tight junctions (TJ) in vertebrates define apical and basolateral membrane domains in polarized epithelia via bi-directional, complex signalling pathways between TJ themselves and the cytoskeleton they are associated with. RhoGTPases are central to these processes and evidence suggests that their regulation is coordinated by interactions between GEFs and GAPs with junctional, cytoplasmic adapter proteins. In this InFocus review we determine that the expression, localization or stability of a variety of these adaptor proteins is altered in various cancers, potentially representing an important mechanistic link between loss of polarity and cancer. We focus here, on two well characterized RhoGTPases Cdc42 and RhoA who's GEFs and GAPs are predominantly localized to TJ via cytoplasmic adaptor proteins. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Methoxychlor and Vinclozolin Induce Rapid Changes in Intercellular and Intracellular Signaling in Liver Progenitor Cells

PubMed Central

Babica, Pavel; Zurabian, Rimma; Kumar, Esha R.; Chopra, Rajus; Mianecki, Maxwell J.; Park, Joon-Suk; Jaša, Libor; Trosko, James E.; Upham, Brad L.

2016-01-01

Methoxychlor (MXC) and vinclozolin (VIN) are well-recognized endocrine disrupting chemicals known to alter epigenetic regulations and transgenerational inheritance; however, non-endocrine disruption endpoints are also important. Thus, we determined the effects of MXC and VIN on the dysregulation of gap junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells. Both chemicals induced a rapid dysregulation of GJIC at non-cytotoxic doses, with 30 min EC50 values for GJIC inhibition being 10 µM for MXC and 126 µM for VIN. MXC inhibited GJIC for at least 24 h, while VIN effects were transient and GJIC recovered after 4 h. VIN induced rapid hyperphosphorylation and internalization of gap junction protein connexin43, and both chemicals also activated MAPK ERK1/2 and p38. Effects on GJIC were not prevented by MEK1/2 inhibitor, but by an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), resveratrol, and in the case of VIN, also, by a p38 inhibitor. Estrogen (ER) and androgen receptor (AR) modulators (estradiol, ICI 182,780, HPTE, testosterone, flutamide, VIN M2) did not attenuate MXC or VIN effects on GJIC. Our data also indicate that the effects were elicited by the parental compounds of MXC and VIN. Our study provides new evidence that MXC and VIN dysregulate GJIC via mechanisms involving rapid activation of PC-PLC occurring independently of ER- or AR-dependent genomic signaling. Such alterations of rapid intercellular and intracellular signaling events involved in regulations of gene expression, tissue development, function and homeostasis, could also contribute to transgenerational epigenetic effects of endocrine disruptors. PMID:27413106

Regulator of G protein signaling 5 (RGS5) inhibits sonic hedgehog function in mouse cortical neurons.

PubMed

Liu, Chuanliang; Hu, Qiongqiong; Jing, Jia; Zhang, Yun; Jin, Jing; Zhang, Liulei; Mu, Lili; Liu, Yumei; Sun, Bo; Zhang, Tongshuai; Kong, Qingfei; Wang, Guangyou; Wang, Dandan; Zhang, Yao; Liu, Xijun; Zhao, Wei; Wang, Jinghua; Feng, Tao; Li, Hulun

2017-09-01

Regulator of G protein signaling 5 (RGS5) acts as a GTPase-activating protein (GAP) for the Gαi subunit and negatively regulates G protein-coupled receptor signaling. However, its presence and function in postmitotic differentiated primary neurons remains largely uncharacterized. During neural development, sonic hedgehog (Shh) signaling is involved in cell signaling pathways via Gαi activity. In particular, Shh signaling is essential for embryonic neural tube patterning, which has been implicated in neuronal polarization involving neurite outgrowth. Here, we examined whether RGS5 regulates Shh signaling in neurons. RGS5 transcripts were found to be expressed in cortical neurons and their expression gradually declined in a time-dependent manner in culture system. When an adenovirus expressing RGS5 was introduced into an in vitro cell culture model of cortical neurons, RGS5 overexpression significantly reduced neurite outgrowth and FM4-64 uptake, while cAMP-PKA signaling was also affected. These findings suggest that RGS5 inhibits Shh function during neurite outgrowth and the presynaptic terminals of primary cortical neurons mature via modulation of cAMP. Copyright © 2017 Elsevier Inc. All rights reserved.

Topography of funneled landscapes determines the thermodynamics and kinetics of protein folding

PubMed Central

Wang, Jin; Oliveira, Ronaldo J.; Chu, Xiakun; Whitford, Paul C.; Chahine, Jorge; Han, Wei; Wang, Erkang; Onuchic, José N.; Leite, Vitor B.P.

2012-01-01

The energy landscape approach has played a fundamental role in advancing our understanding of protein folding. Here, we quantify protein folding energy landscapes by exploring the underlying density of states. We identify three quantities essential for characterizing landscape topography: the stabilizing energy gap between the native and nonnative ensembles δE, the energetic roughness ΔE, and the scale of landscape measured by the entropy S. We show that the dimensionless ratio between the gap, roughness, and entropy of the system accurately predicts the thermodynamics, as well as the kinetics of folding. Large Λ implies that the energy gap (or landscape slope towards the native state) is dominant, leading to more funneled landscapes. We investigate the role of topological and energetic roughness for proteins of different sizes and for proteins of the same size, but with different structural topologies. The landscape topography ratio Λ is shown to be monotonically correlated with the thermodynamic stability against trapping, as characterized by the ratio of folding temperature versus trapping temperature. Furthermore, Λ also monotonically correlates with the folding kinetic rates. These results provide the quantitative bridge between the landscape topography and experimental folding measurements. PMID:23019359

Integrative annotation and knowledge discovery of kinase post-translational modifications and cancer-associated mutations through federated protein ontologies and resources.

PubMed

Huang, Liang-Chin; Ross, Karen E; Baffi, Timothy R; Drabkin, Harold; Kochut, Krzysztof J; Ruan, Zheng; D'Eustachio, Peter; McSkimming, Daniel; Arighi, Cecilia; Chen, Chuming; Natale, Darren A; Smith, Cynthia; Gaudet, Pascale; Newton, Alexandra C; Wu, Cathy; Kannan, Natarajan

2018-04-25

Many bioinformatics resources with unique perspectives on the protein landscape are currently available. However, generating new knowledge from these resources requires interoperable workflows that support cross-resource queries. In this study, we employ federated queries linking information from the Protein Kinase Ontology, iPTMnet, Protein Ontology, neXtProt, and the Mouse Genome Informatics to identify key knowledge gaps in the functional coverage of the human kinome and prioritize understudied kinases, cancer variants and post-translational modifications (PTMs) for functional studies. We identify 32 functional domains enriched in cancer variants and PTMs and generate mechanistic hypotheses on overlapping variant and PTM sites by aggregating information at the residue, protein, pathway and species level from these resources. We experimentally test the hypothesis that S768 phosphorylation in the C-helix of EGFR is inhibitory by showing that oncogenic variants altering S768 phosphorylation increase basal EGFR activity. In contrast, oncogenic variants altering conserved phosphorylation sites in the 'hydrophobic motif' of PKCβII (S660F and S660C) are loss-of-function in that they reduce kinase activity and enhance membrane translocation. Our studies provide a framework for integrative, consistent, and reproducible annotation of the cancer kinomes.

30 CFR 285.652 - How long do I have to conduct activities under an approved GAP?

Code of Federal Regulations, 2010 CFR

2010-07-01

... an approved GAP? 285.652 Section 285.652 Mineral Resources MINERALS MANAGEMENT SERVICE, DEPARTMENT OF... SHELF Plans and Information Requirements Activities Under An Approved Gap § 285.652 How long do I have to conduct activities under an approved GAP? After MMS approves your GAP, you have: (a) For a limited...

30 CFR 285.652 - How long do I have to conduct activities under an approved GAP?

Code of Federal Regulations, 2011 CFR

2011-07-01

... an approved GAP? 285.652 Section 285.652 Mineral Resources BUREAU OF OCEAN ENERGY MANAGEMENT... FACILITIES ON THE OUTER CONTINENTAL SHELF Plans and Information Requirements Activities Under An Approved Gap § 285.652 How long do I have to conduct activities under an approved GAP? After MMS approves your GAP...

GASP: Gapped Ancestral Sequence Prediction for proteins

PubMed Central

Edwards, Richard J; Shields, Denis C

2004-01-01

Background The prediction of ancestral protein sequences from multiple sequence alignments is useful for many bioinformatics analyses. Predicting ancestral sequences is not a simple procedure and relies on accurate alignments and phylogenies. Several algorithms exist based on Maximum Parsimony or Maximum Likelihood methods but many current implementations are unable to process residues with gaps, which may represent insertion/deletion (indel) events or sequence fragments. Results Here we present a new algorithm, GASP (Gapped Ancestral Sequence Prediction), for predicting ancestral sequences from phylogenetic trees and the corresponding multiple sequence alignments. Alignments may be of any size and contain gaps. GASP first assigns the positions of gaps in the phylogeny before using a likelihood-based approach centred on amino acid substitution matrices to assign ancestral amino acids. Important outgroup information is used by first working down from the tips of the tree to the root, using descendant data only to assign probabilities, and then working back up from the root to the tips using descendant and outgroup data to make predictions. GASP was tested on a number of simulated datasets based on real phylogenies. Prediction accuracy for ungapped data was similar to three alternative algorithms tested, with GASP performing better in some cases and worse in others. Adding simple insertions and deletions to the simulated data did not have a detrimental effect on GASP accuracy. Conclusions GASP (Gapped Ancestral Sequence Prediction) will predict ancestral sequences from multiple protein alignments of any size. Although not as accurate in all cases as some of the more sophisticated maximum likelihood approaches, it can process a wide range of input phylogenies and will predict ancestral sequences for gapped and ungapped residues alike. PMID:15350199

A Novel Retinal Oscillation Mechanism in an Autosomal Dominant Photoreceptor Degeneration Mouse Model

PubMed Central

Tu, Hung-Ya; Chen, Yu-Jiun; McQuiston, Adam R.; Chiao, Chuan-Chin; Chen, Ching-Kang

2016-01-01

It has been shown in rd1 and rd10 models of photoreceptor degeneration (PD) that inner retinal neurons display spontaneous and rhythmic activities. Furthermore, the rhythmic activity has been shown to require the gap junction protein connexin 36, which is likely located in AII amacrine cells (AII-ACs). In the present study, an autosomal dominant PD model called rhoΔCTA, whose rods overexpress a C-terminally truncated mutant rhodopsin and degenerate with a rate similar to that of rd1, was used to investigate the generality and mechanisms of heightened inner retinal activity following PD. To fluorescently identify cholinergic starburst amacrine cells (SACs), the rhoΔCTA mouse was introduced into a combined ChAT-IRES-Cre and Ai9 background. In this mouse, we observed excitatory postsynaptic current (EPSC) oscillation and non-rhythmic inhibitory postsynaptic current (IPSC) in both ON- and OFF-SACs. The IPSCs were more noticeable in OFF- than in ON-SACs. Similar to reported retinal ganglion cell (RGC) oscillation in rd1 mice, EPSC oscillation was synaptically driven by glutamate and sensitive to blockade of NaV channels and gap junctions. These data suggest that akin to rd1 mice, AII-AC is a prominent oscillator in rhoΔCTA mice. Surprisingly, OFF-SAC but not ON-SAC EPSC oscillation could readily be enhanced by GABAergic blockade. More importantly, weakening the AII-AC gap junction network by activating retinal dopamine receptors abolished oscillations in ON-SACs but not in OFF-SACs. Furthermore, the latter persisted in the presence of flupirtine, an M-type potassium channel activator recently reported to dampen intrinsic AII-AC bursting. These data suggest the existence of a novel oscillation mechanism in mice with PD. PMID:26793064

Rat Nucleus Accumbens Core Astrocytes Modulate Reward and the Motivation to Self-Administer Ethanol after Abstinence

PubMed Central

Bull, Cecilia; Freitas, Kelen CC; Zou, Shiping; Poland, Ryan S; Syed, Wahab A; Urban, Daniel J; Minter, Sabrina C; Shelton, Keith L; Hauser, Kurt F; Negus, S Stevens; Knapp, Pamela E; Bowers, M Scott

2014-01-01

Our understanding of the active role that astrocytes play in modulating neuronal function and behavior is rapidly expanding, but little is known about the role that astrocytes may play in drug-seeking behavior for commonly abused substances. Given that the nucleus accumbens is critically involved in substance abuse and motivation, we sought to determine whether nucleus accumbens astrocytes influence the motivation to self-administer ethanol following abstinence. We found that the packing density of astrocytes that were expressing glial fibrillary acidic protein increased in the nucleus accumbens core (NAcore) during abstinence from EtOH self-administration. No change was observed in the nucleus accumbens shell. This increased NAcore astrocyte density positively correlated with the motivation for ethanol. Astrocytes can communicate with one another and influence neuronal activity through gap-junction hemichannels. Because of this, the effect of blocking gap-junction hemichannels on the motivation for ethanol was examined. The motivation to self-administer ethanol after 3 weeks abstinence was increased following microinjection of gap-junction hemichannel blockers into the NAcore at doses that block both neuronal and astrocytic channels. In contrast, no effect was observed following microinjection of doses that are not thought to block astrocytic channels or following microinjection of either dose into the nucleus accumbens shell. Additionally, the motivation for sucrose after 3 weeks abstinence was unaffected by NAcore gap-junction hemichannel blockers. Next, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) were selectively expressed in NAcore astrocytes to test the effect of astrocyte stimulation. DREADD activation increased cytosolic calcium in primary astrocytes, facilitated responding for rewarding brain stimulation, and reduced the motivation for ethanol after 3 weeks abstinence. This is the first work to modulate drug-seeking behavior with astrocyte-specific DREADDs. Taken together, our findings demonstrate that NAcore astrocytes can shape the motivation to self-administer ethanol; suggesting that the development of ligands which selectively stimulate astrocytes may be a successful strategy to abate ethanol-seeking behavior. PMID:24903651

Rat nucleus accumbens core astrocytes modulate reward and the motivation to self-administer ethanol after abstinence.

PubMed

Bull, Cecilia; Freitas, Kelen C C; Zou, Shiping; Poland, Ryan S; Syed, Wahab A; Urban, Daniel J; Minter, Sabrina C; Shelton, Keith L; Hauser, Kurt F; Negus, S Stevens; Knapp, Pamela E; Bowers, M Scott

2014-11-01

Our understanding of the active role that astrocytes play in modulating neuronal function and behavior is rapidly expanding, but little is known about the role that astrocytes may play in drug-seeking behavior for commonly abused substances. Given that the nucleus accumbens is critically involved in substance abuse and motivation, we sought to determine whether nucleus accumbens astrocytes influence the motivation to self-administer ethanol following abstinence. We found that the packing density of astrocytes that were expressing glial fibrillary acidic protein increased in the nucleus accumbens core (NAcore) during abstinence from EtOH self-administration. No change was observed in the nucleus accumbens shell. This increased NAcore astrocyte density positively correlated with the motivation for ethanol. Astrocytes can communicate with one another and influence neuronal activity through gap-junction hemichannels. Because of this, the effect of blocking gap-junction hemichannels on the motivation for ethanol was examined. The motivation to self-administer ethanol after 3 weeks abstinence was increased following microinjection of gap-junction hemichannel blockers into the NAcore at doses that block both neuronal and astrocytic channels. In contrast, no effect was observed following microinjection of doses that are not thought to block astrocytic channels or following microinjection of either dose into the nucleus accumbens shell. Additionally, the motivation for sucrose after 3 weeks abstinence was unaffected by NAcore gap-junction hemichannel blockers. Next, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) were selectively expressed in NAcore astrocytes to test the effect of astrocyte stimulation. DREADD activation increased cytosolic calcium in primary astrocytes, facilitated responding for rewarding brain stimulation, and reduced the motivation for ethanol after 3 weeks abstinence. This is the first work to modulate drug-seeking behavior with astrocyte-specific DREADDs. Taken together, our findings demonstrate that NAcore astrocytes can shape the motivation to self-administer ethanol; suggesting that the development of ligands which selectively stimulate astrocytes may be a successful strategy to abate ethanol-seeking behavior.

Deciphering the potential efficacy of acetyl-L-carnitine (ALCAR) in maintaining connexin-mediated lenticular homeostasis

PubMed Central

Muralidharan, Arumugam Ramachandran; Leema, George; Annadurai, Thangaraj; Anitha, Thirugnanasambandhar Sivasubramanian; Thomas, Philip A.

2012-01-01

Purpose To determine the putative role of acetyl-L-carnitine (ALCAR) in maintaining normal intercellular communication in the lens through connexin. Methods In the present study, Wistar rat pups were divided into 3 groups of eight each. On postpartum day ten, Group I rat pups received an intraperitoneal injection (50 µl) of 0.89% saline. Rats in Groups II and III received a subcutaneous injection (50 µl) of sodium selenite (19 µmol/kg bodyweight); Group III rat pups also received an intraperitoneal injection of ALCAR (200 mg/kg bodyweight) once daily on postpartum days 9–14. Both eyes of each pup were examined from day 16 up to postpartum day 30. Alterations in the mean activity of the channel pumps, calcium-ATPase and sodium/potassium-ATPase, were determined. The expression of genes encoding key lenticular gap junctions (connexin 46 and connexin 50) and a channel pump (plasma membrane Ca2+-ATPase [PMCA1]) was evaluated by reverse transcription-PCR. Immunoblot analysis was also performed to confirm the differential expression of key lenticular connexin proteins. In addition, bioinformatics analysis was performed to determine the interacting residues of the connexin proteins with ALCAR. Results Significantly lower mean activities of Ca2+-ATPase and Na+/K+ -ATPase were observed in the lenses of Group II rats than those in Group I rat lenses. However, the observed mean activities of Ca2+-ATPase and Na+/K+-ATPase in Group III rat lenses were significantly higher than those in Group II rat lenses. The mean mRNA transcript levels of the connexin 46 and connexin 50 genes were significantly lower, while the mean levels of PMCA1 gene transcripts were significantly higher, in Group II rat lenses than in Group I rat lenses. Immunoblot analysis also confirmed the altered expression of connexin proteins in lysates of whole lenses of Group II rats. However, the expression of connexin 46 and connexin 50 proteins in lenses from group III rats was essentially similar to that noted in lenses from normal (Group I) rats. Hydrogen bond-interaction between ALCAR and amino acid residues at the functional domain regions of connexin 46 and connexin 50 proteins was also demonstrated through bioinformatics tools. Conclusions The results suggest that ALCAR plays a key role in maintaining lenticular homeostasis by promoting gap junctional intercellular communication. PMID:22876134

Deciphering the potential efficacy of acetyl-L-carnitine (ALCAR) in maintaining connexin-mediated lenticular homeostasis.

PubMed

Muralidharan, Arumugam Ramachandran; Leema, George; Annadurai, Thangaraj; Anitha, Thirugnanasambandhar Sivasubramanian; Thomas, Philip A; Geraldine, Pitchairaj

2012-01-01

To determine the putative role of acetyl-L-carnitine (ALCAR) in maintaining normal intercellular communication in the lens through connexin. In the present study, Wistar rat pups were divided into 3 groups of eight each. On postpartum day ten, Group I rat pups received an intraperitoneal injection (50 µl) of 0.89% saline. Rats in Groups II and III received a subcutaneous injection (50 µl) of sodium selenite (19 µmol/kg bodyweight); Group III rat pups also received an intraperitoneal injection of ALCAR (200 mg/kg bodyweight) once daily on postpartum days 9-14. Both eyes of each pup were examined from day 16 up to postpartum day 30. Alterations in the mean activity of the channel pumps, calcium-ATPase and sodium/potassium-ATPase, were determined. The expression of genes encoding key lenticular gap junctions (connexin 46 and connexin 50) and a channel pump (plasma membrane Ca(2+)-ATPase [PMCA1]) was evaluated by reverse transcription-PCR. Immunoblot analysis was also performed to confirm the differential expression of key lenticular connexin proteins. In addition, bioinformatics analysis was performed to determine the interacting residues of the connexin proteins with ALCAR. Significantly lower mean activities of Ca(2+)-ATPase and Na(+)/K(+) -ATPase were observed in the lenses of Group II rats than those in Group I rat lenses. However, the observed mean activities of Ca(2+)-ATPase and Na(+)/K(+)-ATPase in Group III rat lenses were significantly higher than those in Group II rat lenses. The mean mRNA transcript levels of the connexin 46 and connexin 50 genes were significantly lower, while the mean levels of PMCA1 gene transcripts were significantly higher, in Group II rat lenses than in Group I rat lenses. Immunoblot analysis also confirmed the altered expression of connexin proteins in lysates of whole lenses of Group II rats. However, the expression of connexin 46 and connexin 50 proteins in lenses from group III rats was essentially similar to that noted in lenses from normal (Group I) rats. Hydrogen bond-interaction between ALCAR and amino acid residues at the functional domain regions of connexin 46 and connexin 50 proteins was also demonstrated through bioinformatics tools. The results suggest that ALCAR plays a key role in maintaining lenticular homeostasis by promoting gap junctional intercellular communication.

Attenuation of TRPV1 by AMG-517 after nerve injury promotes peripheral axonal regeneration in rats.

PubMed

Bai, Juan; Liu, Fu; Wu, Li-Fei; Wang, Ya-Fang; Li, Xia-Qing

2018-01-01

Aims The main objective was to investigate the effects of the transient receptor potential cation channel subfamily V member 1 (TRPV1) on nerve regeneration following sciatic transection injury by functional blockage of TRPV1 using AMG-517, a specific blocker of TRPV1. Methods AMG-517 was injected into the area surrounding ipsilateral lumbar dorsal root ganglia 30 min after unilateral sciatic nerve transection. The number of sciatic axons and the expression of growth-associated protein-43 (GAP-43) and glial fibrillary acidic protein was examined using semithin sections, Western blot, and immunofluorescence analyses. Results Blockage of TRPV1 with AMG-517 markedly promoted axonal regeneration, especially at two weeks after sciatic injury; the number of axons was similar to the uninjured control group. After sciatic nerve transection, expression of glial fibrillary acidic protein was decreased and GAP-43 was increased at the proximal stump. However, the expression of both glial fibrillary acidic protein and GAP-43 increased significantly in AMG-517-treated groups. Conclusions TRPV1 may be an important therapeutic target to promote peripheral nerve regeneration after injury.

Expression of connexin 43 mRNA and protein in developing follicles of prepubertal porcine ovaries

USGS Publications Warehouse

Melton, C.M.; Zaunbrecher, G.M.; Yoshizaki, G.; Patio, R.; Whisnant, S.; Rendon, A.; Lee, V.H.

2001-01-01

A major form of cell-cell communication is mediated by gap junctions, aggregations of intercellular channels composed of connexins (Cxs), which are responsible for exchange of low molecular weight (< 1200 Da) cytosolic materials. These channels are a growing family of related proteins. This study was designed to determine the ontogeny of connexin 43 (Cx43) during early stages of follicular development in prepubertal porcine ovaries. A partial-length (412 base) cDNA clone was obtained from mature porcine ovaries and determined to have 98% identity with published porcine Cx43. Northern blot analysis demonstrated a 4.3-kb mRNA in total RNA isolated from prepubertal and adult porcine ovaries. In-situ hybridization revealed that Cx43 mRNA was detectable in granulosa cells of primary follicles but undetectable in dormant primordial follicles. The intensity of the signal increased with follicular growth and was greatest in the large antral follicles. Immunohistochemical evaluation indicated that Cx43 protein expression correlated with the presence of Cx43 mRNA. These results indicate that substantial amounts of Cx43 are first expressed in granulosa cells following activation of follicular development and that this expression increases throughout follicular growth and maturation. These findings suggest an association between the enhancement of intercellular gap-junctional communication and onset of follicular growth. ?? 2001 Elsevier Science Inc. All rights reserved.

Sensitive detection of surface- and size-dependent direct and indirect band gap transitions in ferritin.

PubMed

Colton, J S; Erickson, S D; Smith, T J; Watt, R K

2014-04-04

Ferritin is a protein nano-cage that encapsulates minerals inside an 8 nm cavity. Previous band gap measurements on the native mineral, ferrihydrite, have reported gaps as low as 1.0 eV and as high as 2.5-3.5 eV. To resolve this discrepancy we have used optical absorption spectroscopy, a well-established technique for measuring both direct and indirect band gaps. Our studies included controls on the protein nano-cage, ferritin with the native ferrihydrite mineral, and ferritin with reconstituted ferrihydrite cores of different sizes. We report measurements of an indirect band gap for native ferritin of 2.140 ± 0.015 eV (579.7 nm), with a direct transition appearing at 3.053 ± 0.005 eV (406.1 nm). We also see evidence of a defect-related state having a binding energy of 0.220 ± 0.010 eV . Reconstituted ferrihydrite minerals of different sizes were also studied and showed band gap energies which increased with decreasing size due to quantum confinement effects. Molecules that interact with the surface of the mineral core also demonstrated a small influence following trends in ligand field theory, altering the native mineral's band gap up to 0.035 eV.

MAP kinase pathways in the yeast Saccharomyces cerevisiae

NASA Technical Reports Server (NTRS)

Gustin, M. C.; Albertyn, J.; Alexander, M.; Davenport, K.; McIntire, L. V. (Principal Investigator)

1998-01-01

A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.

Modification and Functional Inhibition of Regulator of G-Protein Signaling 4 (RGS4) by 4-Hydroxy-2-nonenal

PubMed Central

Monroy, C. Aaron; Doorn, Jonathan A.; Roman, David L.

2015-01-01

Oxidative stress has been implicated as a component of various pathologies including ischemia/reperfusion injury (IRI) and neurodegenerative diseases such as Parkinson's disease (PD) and schizophrenia. Similarly, regulator of G-protein signaling 4 (RGS4) has been implicated as an important player in each of these pathologies. RGS4, like other RGS proteins, is responsible for temporally regulating G-protein coupled receptor signaling by increasing the intrinsic GTPase activity of Gα subunit of the heterotrimeric signaling complex. In this study we evaluated whether modification by 4-hydroxy-2-nonenal (4HNE), a common lipid peroxidation product, inhibits RGS4. Using immunoprecipitation, we first determined RGS4 modification was occurring in cells at concentrations of 4HNE within reported physiological conditions. Following this determination, we evaluated modification of RGS4 by 4HNE by both Western blot and mass spectrometry (MS). Once it was established that covalent modification occurred only on cysteine containing constructs, tryptic digest followed by mass spectrometry analysis revealed modification occurs at cysteine residues 71, 148, and 183. In order to determine the effect 4HNE had on RGS4 activity, a steady-state colorimetric assay was used to analyze the GAP activity of Δ51-RGS4 as well as the cysteine null mutant. From the data, we determined that RGS4 activity can be modulated by 4HNE through modification at cysteine residues similar to previously reported small molecule inhibition of RGS4. PMID:24229325

CYK-4 regulates Rac, but not Rho, during cytokinesis

PubMed Central

Zhuravlev, Yelena; Hirsch, Sophia M.; Jordan, Shawn N.; Dumont, Julien; Shirasu-Hiza, Mimi; Canman, Julie C.

2017-01-01

Cytokinesis is driven by constriction of an actomyosin contractile ring that is controlled by Rho-family small GTPases. Rho, activated by the guanine-nucleotide exchange factor ECT-2, is upstream of both myosin-II activation and diaphanous formin-mediated filamentous actin (f-actin) assembly, which drive ring constriction. The role for Rac and its regulators is more controversial, but, based on the finding that Rac inactivation can rescue cytokinesis failure when the GTPase-activating protein (GAP) CYK-4 is disrupted, Rac activity was proposed to be inhibitory to contractile ring constriction and thus specifically inactivated by CYK-4 at the division plane. An alternative model proposes that Rac inactivation generally rescues cytokinesis failure by reducing cortical tension, thus making it easier for the cell to divide when ring constriction is compromised. In this alternative model, CYK-4 was instead proposed to activate Rho by binding ECT-2. Using a combination of time-lapse in vivo single-cell analysis and Caenorhabditis elegans genetics, our evidence does not support this alternative model. First, we found that Rac disruption does not generally rescue cytokinesis failure: inhibition of Rac specifically rescues cytokinesis failure due to disruption of CYK-4 or ECT-2 but does not rescue cytokinesis failure due to disruption of two other contractile ring components, the Rho effectors diaphanous formin and myosin-II. Second, if CYK-4 regulates cytokinesis through Rho rather than Rac, then CYK-4 inhibition should decrease levels of downstream targets of Rho. Inconsistent with this, we found no change in the levels of f-actin or myosin-II at the division plane when CYK-4 GAP activity was reduced, suggesting that CYK-4 is not upstream of ECT-2/Rho activation. Instead, we found that the rescue of cytokinesis in CYK-4 mutants by Rac inactivation was Cdc42 dependent. Together our data suggest that CYK-4 GAP activity opposes Rac (and perhaps Cdc42) during cytokinesis. PMID:28298491

Closing the gap between glia and neuroblast proliferation.

PubMed

Limmer, Stefanie; Klämbt, Christian

2014-08-11

Reporting in this issue of Developmental Cell, Spéder and Brand (2014) show that gap junctions are required in blood-brain barrier glial cells to reactivate proliferation of quiescent neuroblasts. Gap junctions allow synchronous Ca(2+) waves and control insulin-like protein Dipl6 expression and secretion to trigger neuroblast division. Copyright © 2014 Elsevier Inc. All rights reserved.

Production of mouse monoclonal antibody against Streptococcus dysgalactiae GapC protein and mapping its conserved B-cell epitope.

PubMed

Zhang, Limeng; Zhang, Hua; Fan, Ziyao; Zhou, Xue; Yu, Liquan; Sun, Hunan; Wu, Zhijun; Yu, Yongzhong; Song, Baifen; Ma, Jinzhu; Tong, Chunyu; Zhu, Zhanbo; Cui, Yudong

2015-02-01

Streptococcus dysgalactiae (S. dysgalactiae) GapC protein is a protective antigen that induces partial immunity against S. dysgalactiae infection in animals. To identify the conserved B-cell epitope of S. dysgalactiae GapC, a mouse monoclonal antibody 1E11 (mAb1E11) against GapC was generated and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12). Eleven positive clones recognized by mAb1E11 were identified, most of which matched the consensus motif TGFFAKK. Sequence of the motif exactly matched amino acids 97-103 of the S. dysgalactiae GapC. In addition, the epitope (97)TGFFAKK(103) showed high homology among different streptococcus species. Site-directed mutagenic analysis further confirmed that residues G98, F99, F100 and K103 formed the core of (97)TGFFAKK(103), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1E11. Collectively, the identification of conserved B-cell epitope within S. dysgalactiae GapC highlights the possibility of developing the epitope-based vaccine. Copyright © 2014 Elsevier Ltd. All rights reserved.

Direct antigen presentation and gap junction mediated cross-presentation during apoptosis.

PubMed

Pang, Baoxu; Neijssen, Joost; Qiao, Xiaohang; Janssen, Lennert; Janssen, Hans; Lippuner, Christoph; Neefjes, Jacques

2009-07-15

MHC class I molecules present peptides from endogenous proteins. Ags can also be presented when derived from extracellular sources in the form of apoptotic bodies. Cross-presentation of such Ags by dendritic cells is required for proper CTL responses. The fate of Ags in cells initiated for apoptosis is unclear as is the mechanism of apoptosis-derived Ag transfer into dendritic cells. Here we show that novel Ags can be generated by caspases and be presented by MHC class I molecules of apoptotic cells. Since gap junctions function until apoptotic cells remodel to form apoptotic bodies, transfer and cross-presentation of apoptotic peptides by neighboring and dendritic cells occurs. We thus define a novel phase in classical Ag presentation and cross-presentation by MHC class I molecules: presentation of Ags created by caspase activities in cells in apoptosis.

An investigation into the feasibility of myoglobin-based single-electron transistors

PubMed Central

Li, Debin; Gannett, Peter M.; Lederman, David

2016-01-01

Myoglobin single-electron transistors were investigated using nanometer-gap platinum electrodes fabricated by electromigration at cryogenic temperatures. Apomyoglobin (myoglobin without heme group) was used as a reference. The results suggest single electron transport is mediated by resonant tunneling with the electronic and vibrational levels of the heme group in a single protein. They also represent a proof-of-principle that proteins with redox centers across nanometer-gap electrodes can be utilized to fabricate single-electron transistors. The protein orientation and conformation may significantly affect the conductance of these devices. Future improvements in device reproducibility and yield will require control of these factors. PMID:22972432

Blockade of brain stem gap junctions increases phrenic burst frequency and reduces phrenic burst synchronization in adult rat.

PubMed

Solomon, Irene C; Chon, Ki H; Rodriguez, Melissa N

2003-01-01

Recent investigations have examined the influence of gap junctional communication on generation and modulation of respiratory rhythm and inspiratory motoneuron synchronization in vitro using transverse medullary slice and en bloc brain stem-spinal cord preparations obtained from neonatal (1-5 days postnatal) mice. Gap junction proteins, however, have been identified in both neurons and glia in brain stem regions implicated in respiratory control in both neonatal and adult rodents. Here, we used an in vitro arterially perfused rat preparation to examine the role of gap junctional communication on generation and modulation of respiratory rhythm and inspiratory motoneuron synchronization in adult rodents. We recorded rhythmic inspiratory motor activity from one or both phrenic nerves before and during pharmacological blockade (i.e., uncoupling) of brain stem gap junctions using carbenoxolone (100 microM), 18alpha-glycyrrhetinic acid (25-100 microM), 18beta-glycyrrhetinic acid (25-100 microM), octanol (200-300 microM), or heptanol (200 microM). During perfusion with a gap junction uncoupling agent, we observed an increase in the frequency of phrenic bursts (~95% above baseline frequency; P < 0.001) and a decrease in peak amplitude of integrated phrenic nerve discharge (P < 0.001). The increase in frequency of phrenic bursts resulted from a decrease in both T(I) (P < 0.01) and T(E) (P < 0.01). In addition, the pattern of phrenic nerve discharge shifted from an augmenting discharge pattern to a "bell-shaped" or square-wave discharge pattern in most experiments. Spectral analyses using a fast Fourier transform (FFT) algorithm revealed a reduction in the peak power of both the 40- to 50-Hz peak (corresponding to the MFO) and 90- to 110-Hz peak (corresponding to the HFO) although spurious higher frequency activity (> or =130 Hz) was observed, suggesting an overall loss or reduction in inspiratory-phase synchronization. Although additional experiments are required to identify the specific brain stem regions and cell types (i.e., neurons, glia) mediating the observed modulations in phrenic motor output, these findings suggest that gap junction communication modulates generation of respiratory rhythm and inspiratory motoneuron synchronization in adult rodents in vitro.

The Tea4-PP1 landmark promotes local growth by dual Cdc42 GEF recruitment and GAP exclusion.

PubMed

Kokkoris, Kyriakos; Gallo Castro, Daniela; Martin, Sophie G

2014-05-01

Cell polarization relies on small GTPases, such as Cdc42, which can break symmetry through self-organizing principles, and landmarks that define the axis of polarity. In fission yeast, microtubules deliver the Tea1-Tea4 complex to mark cell poles for growth, but how this complex activates Cdc42 is unknown. Here, we show that ectopic targeting of Tea4 to cell sides promotes the local activation of Cdc42 and cell growth. This activity requires that Tea4 binds the type I phosphatase (PP1) catalytic subunit Dis2 or Sds21, and ectopic targeting of either catalytic subunit is similarly instructive for growth. The Cdc42 guanine-nucleotide-exchange factor Gef1 and the GTPase-activating protein Rga4 are required for Tea4-PP1-dependent ectopic growth. Gef1 is recruited to ectopic Tea4 and Dis2 locations to promote Cdc42 activation. By contrast, Rga4 is locally excluded by Tea4, and its forced colocalization with Tea4 blocks ectopic growth, indicating that Rga4 must be present, but at sites distinct from Tea4. Thus, a Tea4-PP1 landmark promotes local Cdc42 activation and growth both through Cdc42 GEF recruitment and by creating a local trough in a Cdc42 GAP.

In silico study of in vitro GPCR assays by QSAR modeling ...

EPA Pesticide Factsheets

The U.S. EPA is screening thousands of chemicals of environmental interest in hundreds of in vitro high-throughput screening (HTS) assays (the ToxCast program). One goal is to prioritize chemicals for more detailed analyses based on activity in molecular initiating events (MIE) of adverse outcome pathways (AOPs). However, the chemical space of interest for environmental exposure is much wider than this set of chemicals. Thus, there is a need to fill data gaps with in silico methods, and quantitative structure-activity relationships (QSARs) are a proven and cost effective approach to predict biological activity. ToxCast in turn provides relatively large datasets that are ideal for training and testing QSAR models. The overall goal of the study described here was to develop QSAR models to fill the data gaps in a larger environmental database of ~32k structures. The specific aim of the current work was to build QSAR models for 18 G-Protein Coupled Receptor (GPCR) assays, part of the aminergic category. Two QSAR modeling strategies were adopted: classification models were developed to separate chemicals into active/non-active classes, and then regression models were built to predict the potency values of the bioassays for the active chemicals. Multiple software programs were used to calculate constitutional, topological and substructural molecular descriptors from two-dimensional (2D) chemical structures. Model-fitting methods included PLSDA (partial least squares d

The Small Hydrophobic Protein of the Human Respiratory Syncytial Virus Forms Pentameric Ion Channels*

PubMed Central

Gan, Siok-Wan; Tan, Edward; Lin, Xin; Yu, Dejie; Wang, Juejin; Tan, Gregory Ming-Yeong; Vararattanavech, Ardcharaporn; Yeo, Chiew Ying; Soon, Cin Huang; Soong, Tuck Wah; Pervushin, Konstantin; Torres, Jaume

2012-01-01

The small hydrophobic (SH) protein is encoded by the human respiratory syncytial virus. Its absence leads to viral attenuation in the context of whole organisms, and it prevents apoptosis in infected cells. Herein, we have examined the structure of SH protein in detergent micelles and in lipid bilayers, by solution NMR and attenuated total reflection-Fourier transform infrared spectroscopy, respectively. We found that SH protein has a single α-helical transmembrane domain and forms homopentamers in several detergents. In detergent micelles, the transmembrane domain is flanked N-terminally by an α-helix that forms a ring around the lumen of the pore and C-terminally by an extended β-turn. SH protein was found in the plasma membrane of transiently expressing HEK 293 cells, which showed pH-dependent (acid-activated) channel activity. Channel activity was abolished in mutants lacking both native His residues, His22 and His51, but not when either His was present. Herein, we propose that the pentameric model of SH protein presented is a physiologically relevant conformation, albeit probably not the only one, in which SH contributes to RSV infection and replication. Viroporins are short (∼100 amino acids) viral membrane proteins that form oligomers of a defined size, act as proton or ion channels, and in general enhance membrane permeability in the host. However, with some exceptions, their precise biological role of their channel activity is not understood. In general, viroporins resemble poorly specialized proteins but are nevertheless critical for viral fitness. In vivo, viruses lacking viroporins usually exhibit an attenuated or weakened phenotype, altered tropism, and diminished pathological effects. We have chosen to study the SH protein, 64 amino acids long, found in the human respiratory syncytial virus because of the effect of RSV on human health and the lack of adequate antivirals. We show that SH protein forms oligomers that behave as ion channels when activated at low pH. This study adds SH protein to a growing group of viroporins that have been structurally characterized. Although the precise biological role of this pentameric channel is still unknown, this report is nevertheless essential to fill some of the many gaps that exist in the understanding of SH protein function. PMID:22621926

Phospholipase D2 Is Involved in the Formation of Golgi Tubules and ArfGAP1 Recruitment

PubMed Central

Martínez-Martínez, Narcisa; Martínez-Alonso, Emma; Ballesta, José; Martínez-Menárguez, José A.

2014-01-01

Lipids and lipid-modifying enzymes play a key role in the biogenesis, maintenance and fission of transport carriers in the secretory and endocytic pathways. In the present study we demonstrate that phosphatidic acid generated by phospholipase D2 (PLD2) is involved in the formation of Golgi tubules. The main evidence to support this is: 1) inhibitors of phosphatidic acid formation and PLD2 depletion inhibit the formation of tubules containing resident enzymes and regulators of intra-Golgi transport in a low temperature (15°C) model of Golgi tubulation but do not affect brefeldin A-induced tubules, 2) inhibition of PLD2 enzymatic activity and PLD2 depletion in cells cultured under physiological conditions (37°C) induce the formation of tubules specifically containing Golgi matrix proteins, and, 3) over-expression of PLD2 induces the formation of a tubular network. In addition, it was found that the generation of this lipid by the isoenzyme is necessary for ArfGAP1 recruitment to Golgi membranes. These results suggest that both proteins are involved in the molecular mechanisms which drive the formation of different types of Golgi tubules. PMID:25354038

GapA, a potential vaccine candidate antigen against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus).

PubMed

Zhang, Ze; Yu, Angen; Lan, Jiangfeng; Zhang, Hua; Hu, Minqiang; Cheng, Jiewei; Zhao, Lijuan; Lin, Li; Wei, Shun

2017-04-01

Streptococcosis due to the bacterium Streptococcus agalactiae (S. agalactiae) has resulted in enormous economic losses in aquaculture worldwide, especially in the tilapia culture industry. Previously, there were limited vaccines that could be employed against streptococcosis in tilapia. This study aimed to develop a vaccine candidate using the glyceraldehyde-phosphate dehydrogenase protein (GapA) of S. agalactiae encoded by the gapA gene. Tilapia were intraperitoneally injected with PBS, PBS + Freund's adjuvant, PBS + Montanide's adjuvant, GapA + Freund's adjuvant, GapA + Montanide's adjuvant, killed S. agalactiae whole cells (WC)+Freund's adjuvant, or killed S. agalactiae whole cells (WC)+ Montanide's adjuvant. They were then challenged with S. agalactiae, and the relative percentage survival (RPS) was monitored 14 days after the challenge. The highest RPSs were observed in the WC groups, with 76.7% in WC + Freund's adjuvant and 74.4% in WC + Montanide's adjuvant groups; these were followed by the GapA groups, with 63.3% in GapA + Freund's adjuvant and 45.6% in GapA + Montanide's adjuvant groups. The RPS of the PBS group was 0%, and those of PBS + Freund's adjuvant and PBS + Montanide's adjuvant groups were 6.7% and 3.3%, respectively. Additionally, the IgM antibody responses elicited in GapA groups and WC groups were significantly higher than those in PBS groups. Furthermore, the expressions of cytokine (IL-1β and TNF-α) mRNAs in the GapA groups and WC groups were significantly higher than those in the PBS groups. Taken together, these results reveal that the GapA protein is a promising vaccine candidate that could be used to prevent streptococcosis in tilapia. Copyright © 2017 Elsevier Ltd. All rights reserved.

Traction force dynamics predict gap formation in activated endothelium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valent, Erik T.; Nieuw Amerongen, Geerten P. van; Hinsbergh, Victor W.M. van

In many pathological conditions the endothelium becomes activated and dysfunctional, resulting in hyperpermeability and plasma leakage. No specific therapies are available yet to control endothelial barrier function, which is regulated by inter-endothelial junctions and the generation of acto-myosin-based contractile forces in the context of cell-cell and cell-matrix interactions. However, the spatiotemporal distribution and stimulus-induced reorganization of these integral forces remain largely unknown. Traction force microscopy of human endothelial monolayers was used to visualize contractile forces in resting cells and during thrombin-induced hyperpermeability. Simultaneously, information about endothelial monolayer integrity, adherens junctions and cytoskeletal proteins (F-actin) were captured. This revealed a heterogeneousmore » distribution of traction forces, with nuclear areas showing lower and cell-cell junctions higher traction forces than the whole-monolayer average. Moreover, junctional forces were asymmetrically distributed among neighboring cells. Force vector orientation analysis showed a good correlation with the alignment of F-actin and revealed contractile forces in newly formed filopodia and lamellipodia-like protrusions within the monolayer. Finally, unstable areas, showing high force fluctuations within the monolayer were prone to form inter-endothelial gaps upon stimulation with thrombin. To conclude, contractile traction forces are heterogeneously distributed within endothelial monolayers and force instability, rather than force magnitude, predicts the stimulus-induced formation of intercellular gaps. - Highlights: • Endothelial monolayers exert dynamic- and heterogeneous traction forces. • High traction forces correlate with junctional areas and the F-actin cytoskeleton. • Newly formed inter-endothelial gaps are characterized by opposing traction forces. • Force stability is a key feature controlling endothelial permeability.« less

Implanted neural progenitor cells regulate glial reaction to brain injury and establish gap junctions with host glial cells.

PubMed

Talaverón, Rocío; Matarredona, Esperanza R; de la Cruz, Rosa R; Macías, David; Gálvez, Victoria; Pastor, Angel M

2014-04-01

Transplantation of neural stem/progenitor cells (NPCs) in the lesioned brain is able to restore morphological and physiological alterations induced by different injuries. The local microenvironment created at the site of grafting and the communication between grafted and host cells are crucial in the beneficial effects attributed to the NPC implants. We have previously described that NPC transplantation in an animal model of central axotomy restores firing properties and synaptic coverage of lesioned neurons and modulates their trophic factor content. In this study, we aim to explore anatomical relationships between implanted NPCs and host glia that might account for the implant-induced neuroprotective effects. Postnatal rat subventricular zone NPCs were isolated and grafted in adult rats after transection of the medial longitudinal fascicle. Brains were removed and analyzed eight weeks later. Immunohistochemistry for different glial markers revealed that NPC-grafted animals displayed significantly greater microglial activation than animals that received only vehicle injections. Implanted NPCs were located in close apposition to activated microglia and reactive astrocytes. The gap junction protein connexin43 was present in NPCs and glial cells at the lesion site and was often found interposed within adjacent implanted and glial cells. Gap junctions were identified between implanted NPCs and host astrocytes and less frequently between NPCs and microglia. Our results show that implanted NPCs modulate the glial reaction to lesion and establish the possibility of communication through gap junctions between grafted and host glial cells which might be involved in the restorative effects of NPC implants. Copyright © 2014 Wiley Periodicals, Inc.

ELMOD1 Stimulates ARF6-GTP Hydrolysis to Stabilize Apical Structures in Developing Vestibular Hair Cells.

PubMed

Krey, Jocelyn F; Dumont, Rachel A; Wilmarth, Philip A; David, Larry L; Johnson, Kenneth R; Barr-Gillespie, Peter G

2018-01-24

Sensory hair cells require control of physical properties of their apical plasma membranes for normal development and function. Members of the ADP-ribosylation factor (ARF) small GTPase family regulate membrane trafficking and cytoskeletal assembly in many cells. We identified ELMO domain-containing protein 1 (ELMOD1), a guanine nucleoside triphosphatase activating protein (GAP) for ARF6, as the most highly enriched ARF regulator in hair cells. To characterize ELMOD1 control of trafficking, we analyzed mice of both sexes from a strain lacking functional ELMOD1 [roundabout ( rda )]. In rda/rda mice, cuticular plates of utricle hair cells initially formed normally, then degenerated after postnatal day 5; large numbers of vesicles invaded the compromised cuticular plate. Hair bundles initially developed normally, but the cell's apical membrane lifted away from the cuticular plate, and stereocilia elongated and fused. Membrane trafficking in type I hair cells, measured by FM1-43 dye labeling, was altered in rda/rda mice. Consistent with the proposed GAP role for ELMOD1, the ARF6 GTP/GDP ratio was significantly elevated in rda/rda utricles compared with controls, and the level of ARF6-GTP was correlated with the severity of the rda/rda phenotype. These results suggest that conversion of ARF6 to its GDP-bound form is necessary for final stabilization of the hair bundle. SIGNIFICANCE STATEMENT Assembly of the mechanically sensitive hair bundle of sensory hair cells requires growth and reorganization of apical actin and membrane structures. Hair bundles and apical membranes in mice with mutations in the Elmod1 gene degenerate after formation, suggesting that the ELMOD1 protein stabilizes these structures. We show that ELMOD1 is a GTPase-activating protein in hair cells for the small GTP-binding protein ARF6, known to participate in actin assembly and membrane trafficking. We propose that conversion of ARF6 into the GDP-bound form in the apical domain of hair cells is essential for stabilizing apical actin structures like the hair bundle and ensuring that the apical membrane forms appropriately around the stereocilia. Copyright © 2018 the authors 0270-6474/18/380843-15$15.00/0.

Observation of giant conductance fluctuations in a protein

NASA Astrophysics Data System (ADS)

Zhang, Bintian; Song, Weisi; Pang, Pei; Zhao, Yanan; Zhang, Peiming; Csabai, István; Vattay, Gábor; Lindsay, Stuart

2017-12-01

Proteins are insulating molecular solids, yet even those containing easily reduced or oxidized centers can have single-molecule electronic conductances that are too large to account for with conventional transport theories. Here, we report the observation of remarkably high electronic conductance states in an electrochemically inactive protein, the ∼200 kD α V β 3 extracellular domain of human integrin. Large current pulses (up to nA) were observed for long durations (many ms, corresponding to many pC of charge transfer) at large gap (>5 nm) distances in an STM when the protein was bound specifically by a small peptide ligand attached to the electrodes. The effect is greatly reduced when a homologous, weakly binding protein (α 4 β 1) is used as a control. In order to overcome the limitations of the STM, the time- and voltage-dependence of the conductance were further explored using a fixed-gap (5 nm) tunneling junction device that was small enough to trap a single protein molecule at any one time. Transitions to a high conductance (∼nS) state were observed, the protein being ‘on’ for times from ms to tenths of a second. The high-conductance states only occur above ∼100 mV applied bias, and thus are not an equilibrium property of the protein. Nanoamp two-level signals indicate the specific capture of a single molecule in an electrode gap functionalized with the ligand. This offers a new approach to label-free electronic detection of single protein molecules. Electronic structure calculations yield a distribution of energy level spacings that is consistent with a recently proposed quantum-critical state for proteins, in which small fluctuations can drive transitions between localized and band-like electronic states.

Observation of Giant Conductance Fluctuations in a Protein

PubMed Central

Zhang, Bintian; Song, Weisi; Pang, Pei; Zhao, Yanan; Zhang, Peiming; Csabai, István; Vattay, Gábor; Lindsay, Stuart

2017-01-01

Proteins are insulating molecular solids, yet even those containing easily reduced or oxidized centers can have single-molecule electronic conductances that are too large to account for with conventional transport theories. Here, we report the observation of remarkably high electronic conductance states in an electrochemically-inactive protein, the ~200 kD αVβ3 extracelluar domain of human integrin. Large current pulses (up to nA) were observed for long durations (many ms, corresponding to many pC of charge transfer) at large gap (>5nm) distances in an STM when the protein was bound specifically by a small peptide ligand attached to the electrodes. The effect is greatly reduced when a homologous, weakly-binding protein (α4β1) is used as a control. In order to overcome the limitations of the STM, the time- and voltage-dependence of the conductance were further explored using a fixed-gap (5 nm) tunneling junction device that was small enough to trap a single protein molecule at any one time. Transitions to a high conductance (~ nS) state were observed, the protein being “on” for times from ms to tenths of a second. The high-conductance states only occur above ~ 100mV applied bias, and thus are not an equilibrium property of the protein. Nanoamp two-level signals indicate the specific capture of a single molecule in an electrode gap functionalized with the ligand. This offers a new approach to label-free electronic detection of single protein molecules. Electronic structure calculations yield a distribution of energy level spacings that is consistent with a recently proposed quantum-critical state for proteins, in which small fluctuations can drive transitions between localized and band-like electronic states. PMID:29552645

A method for partitioning the information contained in a protein sequence between its structure and function.

PubMed

Possenti, Andrea; Vendruscolo, Michele; Camilloni, Carlo; Tiana, Guido

2018-05-23

Proteins employ the information stored in the genetic code and translated into their sequences to carry out well-defined functions in the cellular environment. The possibility to encode for such functions is controlled by the balance between the amount of information supplied by the sequence and that left after that the protein has folded into its structure. We study the amount of information necessary to specify the protein structure, providing an estimate that keeps into account the thermodynamic properties of protein folding. We thus show that the information remaining in the protein sequence after encoding for its structure (the 'information gap') is very close to what needed to encode for its function and interactions. Then, by predicting the information gap directly from the protein sequence, we show that it may be possible to use these insights from information theory to discriminate between ordered and disordered proteins, to identify unknown functions, and to optimize artificially-designed protein sequences. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

G-LoSA for Prediction of Protein-Ligand Binding Sites and Structures.

PubMed

Lee, Hui Sun; Im, Wonpil

2017-01-01

Recent advances in high-throughput structure determination and computational protein structure prediction have significantly enriched the universe of protein structure. However, there is still a large gap between the number of available protein structures and that of proteins with annotated function in high accuracy. Computational structure-based protein function prediction has emerged to reduce this knowledge gap. The identification of a ligand binding site and its structure is critical to the determination of a protein's molecular function. We present a computational methodology for predicting small molecule ligand binding site and ligand structure using G-LoSA, our protein local structure alignment and similarity measurement tool. All the computational procedures described here can be easily implemented using G-LoSA Toolkit, a package of standalone software programs and preprocessed PDB structure libraries. G-LoSA and G-LoSA Toolkit are freely available to academic users at http://compbio.lehigh.edu/GLoSA . We also illustrate a case study to show the potential of our template-based approach harnessing G-LoSA for protein function prediction.

Up-regulation of GAP-43 (B50/F1) gene expression in vestibular efferent neurons following labyrinthectomy in the rat: in situ hybridization using an alkaline phosphatase-labeled probe.

PubMed

Ohno, K; Takeda, N; Kubo, T; Kiyama, H

1994-10-01

Growth-associated protein (GAP)-43 plays a significant role in nerve regeneration and synaptic remodeling. We examined the profiles of GAP-43 mRNA expression in vestibular efferent neurons after labyrinthectomy in adult rats, and clearly demonstrated that labyrinthectomy increased GAP-43 expression in these neurons. This finding suggests the ability of vestibular efferent nerves to regenerate after nerve injury.

p38 Mitogen Activated Protein Kinase (MAPK): A New Therapeutic Target for Reducing the Risk of Adverse Pregnancy Outcomes

PubMed Central

Menon, Ramkumar; Papaconstantinou, John

2016-01-01

Introduction Spontaneous preterm birth (PTB) and preterm premature rupture of the membranes (pPROM) remain as a major clinical and therapeutic problem for intervention and management. Current strategies, based on our knowledge of pathways of preterm labor, have only been effective, in part, due to major gaps in our existing knowledge of risks and risk specific pathways. Areas covered Recent literature has identified physiologic aging of fetal tissues as a potential mechanistic feature of normal parturition. This process is affected by telomere dependent and p38 mitogen activated protein kinase (MAPK) induced senescence activation. Pregnancy associated risk factors can cause pathologic activation of this pathway that can cause oxidative stress induced p38 MAPK activation leading to senescence and premature aging of fetal tissues. Premature aging is associated with sterile inflammation capable of triggering preterm labor or preterm premature rupture of membranes. Preterm activation of p38MAPK can be considered as a key contributor to adverse pregnancies. Expert Opinion This review considers p38MAPK activation as a potential target for therapeutic interventions to prevent adverse pregnancy outcomes mediated by stress factors. In this review, we propose multiple strategies to prevent p38MAPK activation and its functional effects. PMID:27459026

RACK1 promotes radiation resistance in esophageal cancer via regulating AKT pathway and Bcl-2 expression.

PubMed

Liu, Bowen; Wang, Cong; Chen, Pengxiang; Wang, Lu; Cheng, Yufeng

2017-09-23

RACK1 is a seven Trp-Asp 40 repeat protein, which interacts with a wide range of kinases and proteins. RACK1 plays an important role in the proliferation and progression of various cancers. The aim of this study is to detect the role of RACK1 in the radioresistance in esophageal cancer. The results indicated that downregulation of RACK1 reduced the colony formation ability, proliferation ability and resistance of cells to radiation effection through regulating the radiation-related proteins including pAKT, Bcl-2 and Bim; whereas upregulation of RACK1 promoted the ability and radioresistance of ESCC cells. Our findings suggest that RACK1 promotes proliferation and radioresistance in ESCC cells by activating the AKT pathway, upregulating Bcl-2 expression and downregulating protein levels of Bim. Our study fills in gaps in the field of RACK1 and radiation resistance and may provide new possibilities for improving strategies of radiotherapy in esophageal cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Genetic and functional analysis of the gene encoding GAP-43 in schizophrenia.

PubMed

Shen, Yu-Chih; Tsai, Ho-Min; Cheng, Min-Chih; Hsu, Shih-Hsin; Chen, Shih-Fen; Chen, Chia-Hsiang

2012-02-01

In earlier reports, growth-associated protein 43 (GAP-43) has been shown to be critical for initial establishment or reorganization of synaptic connections, a process thought to be disrupted in schizophrenia. Additionally, abnormal GAP-43 expression in different brain regions has been linked to this disorder in postmortem brain studies. In this study, we investigated the involvement of the gene encoding GAP-43 in the susceptibility to schizophrenia. We searched for genetic variants in the promoter region and 3 exons (including both UTR ends) of the GAP-43 gene using direct sequencing in a sample of patients with schizophrenia (n=586) and non-psychotic controls (n=576), both being Han Chinese from Taiwan, and conducted an association and functional study. We identified 11 common polymorphisms in the GAP-43 gene. SNP and haplotype-based analyses displayed no associations with schizophrenia. Additionally, we identified 4 rare variants in 5 out of 586 patients, including 1 variant located at the promoter region (c.-258-4722G>T) and 1 synonymous (V110V) and 2 missense (G150R and P188L) variants located at exon 2. No rare variants were found in the control subjects. The results of the reporter gene assay demonstrated that the regulatory activity of construct containing c.-258-4722T was significantly lower as compared to the wild type construct (c.-258-4722G; p<0.001). In silico analysis also demonstrated the functional relevance of other rare variants. Our study lends support to the hypothesis of multiple rare mutations in schizophrenia, and it provides genetic clues that indicate the involvement of GAP-43 in this disorder. Copyright © 2011 Elsevier B.V. All rights reserved.

Structure of the Integral Membrane Protein CAAX Protease Ste24p

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pryor Jr., Edward E.; Horanyi, Peter S.; Clark, Kathleen M.

2012-10-26

Posttranslational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CAAX sequence motifs (where A is an aliphatic residue and X is any residue). Isoprenylation is followed by cleavage of the AAX amino acid residues and, in some cases, by additional proteolytic cuts. We determined the crystal structure of the CAAX protease Ste24p, a zinc metalloprotease catalyzing two proteolytic steps in the maturation of yeast mating pheromone a -factor. The Ste24p core structure is a ring of seven transmembrane helices enclosing a voluminous cavitymore » containing the active site and substrate-binding groove. The cavity is accessible to the external milieu by means of gaps between splayed transmembrane helices. We hypothesize that cleavage proceeds by means of a processive mechanism of substrate insertion, translocation, and ejection.« less

Physiological Role of Gap-Junctional Hemichannels

PubMed Central

Quist, Arjan Pieter; Rhee, Seung Keun; Lin, Hai; Lal, Ratneshwar

2000-01-01

Hemichannels in the overlapping regions of apposing cells plasma membranes join to form gap junctions and provide an intercellular communication pathway. Hemichannels are also present in the nonjunctional regions of individual cells and their activity is gated by several agents, including calcium. However, their physiological roles are unknown. Using techniques of atomic force microscopy (AFM), fluorescent dye uptake assay, and laser confocal immunofluorescence imaging, we have examined the extracellular calcium-dependent modulation of cell volume. In response to a change in the extracellular physiological calcium concentration (1.8 to ≤1.6 mM) in an otherwise isosmotic condition, real-time AFM imaging revealed a significant and reversible increase in the volume of cells expressing gap-junctional proteins (connexins). Volume change did not occur in cells that were not expressing connexins. However, after the transient or stable transfection of connexin43, volume change did occur. The volume increase was accompanied by cytochalasin D-sensitive higher cell stiffness, which helped maintain cell integrity. These cellular physical changes were prevented by gap-junctional blockers, oleamide and β-glycyrrhetinic acid, or were reversed by returning extracellular calcium to the normal level. We conclude that nongap-junctional hemichannels regulate cell volume in response to the change in extracellular physiological calcium in an otherwise isosmotic situation. PMID:10704454

Sestrin2 inhibits mTORC1 through modulation of GATOR complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jeong Sig; Ro, Seung-Hyun; Kim, Myungjin

2015-03-30

Sestrins are stress-inducible metabolic regulators that suppress a wide range of age- and obesity-associated pathologies, many of which are due to mTORC1 overactivation. Upon various stresses, the Sestrins inhibit mTORC1 activity through an indirect mechanism that is still unclear. GATORs are recently identified protein complexes that regulate the activity of RagB, a small GTPase essential for mTORC1 activation. GATOR1 is a GTPase activating protein (GAP) for RagB whereas GATOR2 functions as an inhibitor of GATOR1. However, how the GATORs are physiologically regulated is unknown. Here we show that Sestrin2 binds to GATOR2, and liberates GATOR1 from GATOR2-mediated inhibition. Released GATOR1more » subsequently binds to and inactivates RagB, ultimately resulting in mTORC1 suppression. Consistent with this biochemical mechanism, genetic ablation of GATOR1 nullifies the mTORC1-inhibiting effect of Sestrin2 in both cell culture and Drosophila models. Collectively, we elucidate a new signaling cascade composed of Sestrin2-GATOR2-GATOR1-RagB that mediates stress-dependent suppression of mTORC1 activity.« less

Semantic-gap-oriented active learning for multilabel image annotation.

PubMed

Tang, Jinhui; Zha, Zheng-Jun; Tao, Dacheng; Chua, Tat-Seng

2012-04-01

User interaction is an effective way to handle the semantic gap problem in image annotation. To minimize user effort in the interactions, many active learning methods were proposed. These methods treat the semantic concepts individually or correlatively. However, they still neglect the key motivation of user feedback: to tackle the semantic gap. The size of the semantic gap of each concept is an important factor that affects the performance of user feedback. User should pay more efforts to the concepts with large semantic gaps, and vice versa. In this paper, we propose a semantic-gap-oriented active learning method, which incorporates the semantic gap measure into the information-minimization-based sample selection strategy. The basic learning model used in the active learning framework is an extended multilabel version of the sparse-graph-based semisupervised learning method that incorporates the semantic correlation. Extensive experiments conducted on two benchmark image data sets demonstrated the importance of bringing the semantic gap measure into the active learning process.

A Direct Redox Regulation of Protein Kinase C Isoenzymes Mediates Oxidant-induced Neuritogenesis in PC12 Cells*

PubMed Central

Gopalakrishna, Rayudu; Gundimeda, Usha; Schiffman, Jason Eric; McNeill, Thomas H.

2008-01-01

In this study, we have used the PC12 cell model to elucidate the mechanisms by which sublethal doses of oxidants induce neuritogenesis. The xanthine/xanthine oxidase (X/XO) system was used for the steady state generation of superoxide, and CoCl2 was used as a representative transition metal redox catalyst. Upon treatment of purified protein kinase C (PKC) with these oxidants, there was an increase in its cofactor-independent activation. Redox-active cobalt competed with the redoxinert zinc present in the zinc-thiolates of the PKC regulatory domain and induced the oxidation of these cysteine-rich regions. Both CoCl2 and X/XO induced neurite outgrowth in PC12 cells, as determined by an overexpression of neuronal marker genes. Furthermore, these oxidants induced a translocation of PKC from cytosol to membrane and subsequent conversion of PKC to a cofactor-independent form. Isoenzyme-specific PKC inhibitors demonstrated that PKCε plays a crucial role in neuritogenesis. Moreover, oxidant-induced neurite outgrowth was increased with a conditional overexpression of PKCε and decreased with its knock-out by small interfering RNA. Parallel with PKC activation, an increase in phosphorylation of the growth-associated neuronal protein GAP-43 at Ser41 was observed. Additionally, there was a sustained activation of extracellular signal-regulated kinases 1 and 2, which was correlated with activating phosphorylation (Ser133) of cAMP-responsive element-binding protein. All of these signaling events that are causally linked to neuritogenesis were blocked by antioxidant N-acetylcysteine (both l and d-forms) and by a variety of PKC-specific inhibitors. Taken together, these results strongly suggest that sublethal doses of oxidants induce neuritogenesis via a direct redox activation of PKCε. PMID:18375950

Early structural changes of the heart after experimental polytrauma and hemorrhagic shock

PubMed Central

Halbgebauer, Rebecca; Eisele, Philipp; Messerer, David A. C.; Weckbach, Sebastian; Schultze, Anke; Braumüller, Sonja; Gebhard, Florian

2017-01-01

Evidence is emerging that systemic inflammation after trauma drives structural and functional impairment of cardiomyocytes and leads to cardiac dysfunction, thus worsening the outcome of polytrauma patients. This study investigates the structural and molecular changes in heart tissue 4 h after multiple injuries with additional hemorrhagic shock using a clinically relevant rodent model of polytrauma. We determined mediators of systemic inflammation (keratinocyte chemoattractant, macrophage chemotactic protein 1), activated complement component C3a and cardiac troponin I in plasma and assessed histological specimen of the mouse heart via standard histomorphology and immunohistochemistry for cellular and subcellular damage and ongoing apoptosis. Further we investigated spatial and quantitative changes of connexin 43 by immunohistochemistry and western blotting. Our results show significantly increased plasma levels of both keratinocyte chemoattractant and cardiac troponin I 4 h after polytrauma and 2 h after induction of hypovolemia. Although we could not detect any morphological changes, immunohistochemical evaluation showed increased level of tissue high-mobility group box 1, which is both a damage-associated molecule and actively released as a danger response signal. Additionally, there was marked lateralization of the cardiac gap-junction protein connexin 43 following combined polytrauma and hemorrhagic shock. These results demonstrate a molecular manifestation of remote injury of cardiac muscle cells in the early phase after polytrauma and hemorrhagic shock with marked disruption of the cardiac gap junction. This disruption of an important component of the electrical conduction system of the heart may lead to arrhythmia and consequently to cardiac dysfunction. PMID:29084268

Pannexins and gap junction protein diversity.

PubMed

Shestopalov, V I; Panchin, Y

2008-02-01

Gap junctions (GJs) are composed of proteins that form a channel connecting the cytoplasm of adjacent cells. Connexins were initially considered to be the only proteins capable of GJ formation. Another family of GJ proteins (innexins) were first found in invertebrates and were proposed to be renamed pannexins after their orthologs were discovered in vertebrates. The lack of both connexins and pannexins in the genomes of some metazoans suggests that other, still undiscovered GJ proteins exist. In vertebrates, connexins and pannexins co-exist. Here we discuss whether vertebrate pannexins have a nonredundant role in animal physiology. Pannexin channels appear to be suited for ATP and calcium signaling and play a role in the maintenance of calcium homeostasis by mechanisms implicating both GJ and nonjunctional function. Suggested roles in the ischemic death of neurons, schizophrenia, inflammation and tumor suppression have drawn much attention to exploring the molecular properties and cellular functions of pannexins.

DNATagger, colors for codons.

PubMed

Scherer, N M; Basso, D M

2008-09-16

DNATagger is a web-based tool for coloring and editing DNA, RNA and protein sequences and alignments. It is dedicated to the visualization of protein coding sequences and also protein sequence alignments to facilitate the comprehension of evolutionary processes in sequence analysis. The distinctive feature of DNATagger is the use of codons as informative units for coloring DNA and RNA sequences. The codons are colored according to their corresponding amino acids. It is the first program that colors codons in DNA sequences without being affected by "out-of-frame" gaps of alignments. It can handle single gaps and gaps inside the triplets. The program also provides the possibility to edit the alignments and change color patterns and translation tables. DNATagger is a JavaScript application, following the W3C guidelines, designed to work on standards-compliant web browsers. It therefore requires no installation and is platform independent. The web-based DNATagger is available as free and open source software at http://www.inf.ufrgs.br/~dmbasso/dnatagger/.

Rapid Modulation of Protein Expression in the Rat Hippocampus Following Deep Brain Stimulation of the Fornix.

PubMed

Gondard, Elise; Chau, Hien N; Mann, Amandeep; Tierney, Travis S; Hamani, Clement; Kalia, Suneil K; Lozano, Andres M

2015-01-01

The forniceal area is currently being evaluated as a target for deep brain stimulation (DBS) to improve cognitive function in patients with Alzheimer's disease. The molecular changes at downstream targets within the stimulated circuit are unknown. To analyze the modulation of hippocampal protein expression following 1 h of fornix DBS in the rat. Animals underwent bilateral forniceal DBS for 1 h and sacrificed at different time-points after the initiation of the stimulation (1 h, 2.5 h, 5 h, 25 h). Bilateral hippocampi were isolated for western blot analyses. Forniceal DBS led to a dramatic elevation of cFos post-stimulation, suggesting that forniceal DBS activates the hippocampus. There was also a significant increase in candidate proteins including several trophic factors, such as brain derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) but not glial cell-derived neurotrophic factor (GDNF). There was in addition, increased expression of the synaptic markers growth associated protein 43 (GAP-43), synaptophysin and α-synuclein. No changes were observed at the studied time-points in Alzheimer's-related proteins including amyloid precursor protein (APP), tau, phosphorylated tau (ptau), or selected chaperone proteins (HSP40, HSP70 and CHIP). Forniceal DBS triggers hippocampal activity and rapidly modulate the expression of neurotrophic factors and markers of synaptic plasticity known to play key roles in memory processing. The clinical effects of DBS of the fornix may, in part, be mediated by producing changes in the expression of these proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of multipurpose solutions (MPS) for hydrogel contact lenses on gap-junctional intercellular communication (GJIC) in rabbit corneal keratocytes.

PubMed

Sumide, Taizo; Tsuchiya, Toshie

2003-02-15

To ensure the effects of multipurpose solutions (MPS) for hydrogel contact lenses on the cornea, the inhibitory activity of three types of MPS on corneal cells has been evaluated with the use of scrape loading and dye transfer assay (SLDT assay) and Western blotting on rabbit corneal keratocytes (RC4). In SLDT assay, MPS-A and poloxamine showed dose-dependent inhibitory activity, suggesting the inhibitory action of MPS-A and poloxamine to gap junctional intercellular communication (GJIC) in the tested cells. Moreover, after treatment with MPS-A, the GJIC was initially inhibited within 4 h, and thereafter gradually turned to an approximately 60% level of the initial value. When MPS-A was removed from the incubation media after exposure of the cells, the recovery of GJIC was time dependent and returned to approximately initial levels at 8 h. Complete recovery was established after approximately 24 h. These findings suggested that the inhibitory action of MPS-A on corneal keratocytes was reversible. This inhibition was accompanied by a decrease in the quantity of connexin-43, which is a major protein constituting the gap junctional channel of these cells, and its change in the phosphorylation state. Taken together, it was suggested that MPS-A interacts with connexin-43, inducing an inhibitory action on GJIC. (c) 2002 Wiley Periodicals, Inc.

A HuD-ZBP1 ribonucleoprotein complex localizes GAP-43 mRNA into axons through its 3' untranslated region AU-rich regulatory element.

PubMed

Yoo, Soonmoon; Kim, Hak H; Kim, Paul; Donnelly, Christopher J; Kalinski, Ashley L; Vuppalanchi, Deepika; Park, Michael; Lee, Seung J; Merianda, Tanuja T; Perrone-Bizzozero, Nora I; Twiss, Jeffery L

2013-09-01

Localized translation of axonal mRNAs contributes to developmental and regenerative axon growth. Although untranslated regions (UTRs) of many different axonal mRNAs appear to drive their localization, there has been no consensus RNA structure responsible for this localization. We recently showed that limited expression of ZBP1 protein restricts axonal localization of both β-actin and GAP-43 mRNAs. β-actin 3'UTR has a defined element for interaction with ZBP1, but GAP-43 mRNA shows no homology to this RNA sequence. Here, we show that an AU-rich regulatory element (ARE) in GAP-43's 3'UTR is necessary and sufficient for its axonal localization. Axonal GAP-43 mRNA levels increase after in vivo injury, and GAP-43 mRNA shows an increased half-life in regenerating axons. GAP-43 mRNA interacts with both HuD and ZBP1, and HuD and ZBP1 co-immunoprecipitate in an RNA-dependent fashion. Reporter mRNA with the GAP-43 ARE competes with endogenous β-actin mRNA for axonal localization and decreases axon length and branching similar to the β-actin 3'UTR competing with endogenous GAP-43 mRNA. Conversely, over-expressing GAP-43 coding sequence with its 3'UTR ARE increases axonal elongation and this effect is lost when just the ARE is deleted from GAP-43's 3'UTR. We have recently found that over-expression of GAP-43 using an axonally targeted construct with the 3'UTRs of GAP-43 promoted elongating growth of axons, while restricting the mRNA to the cell body with the 3'UTR of γ-actin had minimal effect on axon length. In this study, we show that the ARE in GAP-43's 3'UTR is responsible for localization of GAP-43 mRNA into axons and is sufficient for GAP-43 protein's role in elongating axonal growth. © 2013 International Society for Neurochemistry.

Reconstitution of the NF1 GAP-related domain in NF1-deficient human Schwann cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thomas, Stacey L.; Neuroscience Program, Loyola University Medical Center, Maywood, IL; Department of Anatomy and Cell Biology, University of Illinois Chicago, Chicago, IL

Schwann cells derived from peripheral nerve sheath tumors from individuals with Neurofibromatosis Type 1 (NF1) are deficient for the protein neurofibromin, which contains a GAP-related domain (NF1-GRD). Neurofibromin-deficient Schwann cells have increased Ras activation, increased proliferation in response to certain growth stimuli, increased angiogenic potential, and altered cell morphology. This study examined whether expression of functional NF1-GRD can reverse the transformed phenotype of neurofibromin-deficient Schwann cells from both benign and malignant peripheral nerve sheath tumors. We reconstituted the NF1-GRD using retroviral transduction and examined the effects on cell morphology, growth potential, and angiogenic potential. NF1-GRD reconstitution resulted in morphologic changes,more » a 16-33% reduction in Ras activation, and a 53% decrease in proliferation in neurofibromin-deficient Schwann cells. However, NF1-GRD reconstitution was not sufficient to decrease the in vitro angiogenic potential of the cells. This study demonstrates that reconstitution of the NF1-GRD can at least partially reverse the transformation of human NF1 tumor-derived Schwann cells.« less

Immunogenicity of Recombinant Proteins Consisting of Plasmodium vivax Circumsporozoite Protein Allelic Variant-Derived Epitopes Fused with Salmonella enterica Serovar Typhimurium Flagellin

PubMed Central

Leal, Monica Teixeira Andrade; Camacho, Ariane Guglielmi Ariza; Teixeira, Laís Helena; Bargieri, Daniel Youssef; Soares, Irene Silva; Tararam, Cibele Aparecida

2013-01-01

A Plasmodium falciparum circumsporozoite protein (CSP)-based recombinant fusion vaccine is the first malaria vaccine to reach phase III clinical trials. Resistance to infection correlated with the production of antibodies to the immunodominant central repeat region of the CSP. In contrast to P. falciparum, vaccine development against the CSP of Plasmodium vivax malaria is far behind. Based on this gap in our knowledge, we generated a recombinant chimeric protein containing the immunodominant central repeat regions of the P. vivax CSP fused to Salmonella enterica serovar Typhimurium-derived flagellin (FliC) to activate the innate immune system. The recombinant proteins that were generated contained repeat regions derived from each of the 3 different allelic variants of the P. vivax CSP or a fusion of regions derived from each of the 3 allelic forms. Mice were subcutaneously immunized with the fusion proteins alone or in combination with the Toll-like receptor 3 (TLR-3) agonist poly(I·C), and the anti-CSP serum IgG response was measured. Immunization with a mixture of the 3 recombinant proteins, each containing immunodominant epitopes derived from a single allelic variant, rather than a single recombinant protein carrying a fusion of regions derived from each of 3 allelic forms elicited a stronger immune response. This response was independent of TLR-4 but required TLR-5/MyD88 activation. Antibody titers significantly increased when poly(I·C) was used as an adjuvant with a mixture of the 3 recombinant proteins. These recombinant fusion proteins are novel candidates for the development of an effective malaria vaccine against P. vivax. PMID:23863502

Proteomic analysis of protein interactions between Eimeria maxima sporozoites and chicken jejunal epithelial cells by shotgun LC-MS/MS.

PubMed

Huang, Jingwei; Liu, Tingqi; Li, Ke; Song, Xiaokai; Yan, Ruofeng; Xu, Lixin; Li, Xiangrui

2018-04-04

Eimeria maxima initiates infection by invading the jejunal epithelial cells of chicken. However, the proteins involved in invasion remain unknown. The research of the molecules that participate in the interactions between E. maxima sporozoites and host target cells will fill a gap in our understanding of the invasion system of this parasitic pathogen. In the present study, chicken jejunal epithelial cells were isolated and cultured in vitro. Western blot was employed to analyze the soluble proteins of E. maxima sporozoites that bound to chicken jejunal epithelial cells. Co-immunoprecipitation (co-IP) assay was used to separate the E. maxima proteins that bound to chicken jejunal epithelial cells. Shotgun LC-MS/MS technique was used for proteomics identification and Gene Ontology was employed for the bioinformatics analysis. The results of Western blot analysis showed that four proteins bands from jejunal epithelial cells co-cultured with soluble proteins of E. maxima sporozoites were recognized by the positive sera, with molecular weights of 70, 90, 95 and 130 kDa. The co-IP dilutions were analyzed by shotgun LC-MS/MS. A total of 204 proteins were identified in the E. maxima protein database using the MASCOT search engine. Thirty-five proteins including microneme protein 3 and 7 had more than two unique peptide counts and were annotated using Gene Ontology for molecular function, biological process and cellular localization. The results revealed that of the 35 annotated peptides, 22 (62.86%) were associated with binding activity and 15 (42.86%) were involved in catalytic activity. Our findings provide an insight into the interaction between E. maxima and the corresponding host cells and it is important for the understanding of molecular mechanisms underlying E. maxima invasion.

Effects of ageing and streptozotocin–induced diabetes on connexin43 and P2 purinoceptor expression in the rat corpora cavernosa and urinary bladder

PubMed Central

Suadicani, Sylvia O.; Urban–Maldonado, Marcia; Tar, Moses T.; Melman, Arnold; Spray, David C.

2012-01-01

OBJECTIVE To investigate whether ageing and diabetes alter the expression of the gap junction protein connexin43 (Cx43) and of particular purinoceptor (P2R) subtypes in the corpus cavernosum and urinary bladder, and determine whether changes in expression of these proteins correlate with development of erectile and bladder dysfunction in diabetic and ageing rats. MATERIALS AND METHODS Erectile and bladder function of streptozotocin (STZ)-induced diabetic, insulin-treated and age-matched control Fischer-344 rats were evaluated 2, 4 and 8 months after diabetes induction by in vivo cystometry and cavernosometry. Corporal and bladder tissue were then isolated at each of these sample times and protein expression levels of Cx43 and of various P2R subtypes were determined by Western blotting. RESULTS In the corpora of control rats ageing was accompanied by a significant decrease in Cx43 and P2X1R, and increase in P2X7R expression. There was decreased Cx43 and increased P2Y4R expression in the ageing control rat bladder. There was a significant negative correlation between erectile capacity and P2X1R expression levels, and a positive correlation between bladder spontaneous activity and P2Y4R expression levels. There was already development of erectile dysfunction and bladder overactivity at 2 months after inducing diabetes, the earliest sample measured in the study. The development of these urogenital complications was accompanied by significant decreases in Cx43, P2Y2R, P2X4R and increase in P2X1R expression in the corpora, and by a doubling in Cx43 and P2Y2R, and significant increase in P2Y4R expression in the bladder. Changes in Cx43 and P2R expression were largely prevented by insulin therapy. CONCLUSION Ageing and diabetes mellitus markedly altered the expression of the gap junction protein Cx43 and of particular P2R subtypes in the rat penile corpora and urinary bladder. These changes in Cx43 and P2R expression provide the molecular substrate for altered gap junction and purinergic signalling in these tissues, and thus probably contribute to the early development of erectile dysfunction and higher detrusor activity in ageing and in diabetic rats. PMID:19154470

An Augmented Pocketome: Detection and Analysis of Small-Molecule Binding Pockets in Proteins of Known 3D Structure.

PubMed

Bhagavat, Raghu; Sankar, Santhosh; Srinivasan, Narayanaswamy; Chandra, Nagasuma

2018-03-06

Protein-ligand interactions form the basis of most cellular events. Identifying ligand binding pockets in proteins will greatly facilitate rationalizing and predicting protein function. Ligand binding sites are unknown for many proteins of known three-dimensional (3D) structure, creating a gap in our understanding of protein structure-function relationships. To bridge this gap, we detect pockets in proteins of known 3D structures, using computational techniques. This augmented pocketome (PocketDB) consists of 249,096 pockets, which is about seven times larger than what is currently known. We deduce possible ligand associations for about 46% of the newly identified pockets. The augmented pocketome, when subjected to clustering based on similarities among pockets, yielded 2,161 site types, which are associated with 1,037 ligand types, together providing fold-site-type-ligand-type associations. The PocketDB resource facilitates a structure-based function annotation, delineation of the structural basis of ligand recognition, and provides functional clues for domains of unknown functions, allosteric proteins, and druggable pockets. Copyright © 2018 Elsevier Ltd. All rights reserved.

Advances in Homology Protein Structure Modeling

PubMed Central

Xiang, Zhexin

2007-01-01

Homology modeling plays a central role in determining protein structure in the structural genomics project. The importance of homology modeling has been steadily increasing because of the large gap that exists between the overwhelming number of available protein sequences and experimentally solved protein structures, and also, more importantly, because of the increasing reliability and accuracy of the method. In fact, a protein sequence with over 30% identity to a known structure can often be predicted with an accuracy equivalent to a low-resolution X-ray structure. The recent advances in homology modeling, especially in detecting distant homologues, aligning sequences with template structures, modeling of loops and side chains, as well as detecting errors in a model, have contributed to reliable prediction of protein structure, which was not possible even several years ago. The ongoing efforts in solving protein structures, which can be time-consuming and often difficult, will continue to spur the development of a host of new computational methods that can fill in the gap and further contribute to understanding the relationship between protein structure and function. PMID:16787261

The C1 domain-targeted isophthalate derivative HMI-1b11 promotes neurite outgrowth and GAP-43 expression through PKCα activation in SH-SY5Y cells.

PubMed

Talman, Virpi; Amadio, Marialaura; Osera, Cecilia; Sorvari, Salla; Boije Af Gennäs, Gustav; Yli-Kauhaluoma, Jari; Rossi, Daniela; Govoni, Stefano; Collina, Simona; Ekokoski, Elina; Tuominen, Raimo K; Pascale, Alessia

2013-07-01

Protein kinase C (PKC) is a family of serine/threonine phosphotransferases ubiquitously expressed and involved in multiple cellular functions, such as proliferation, apoptosis and differentiation. The C1 domain of PKC represents an attractive drug target, especially for developing PKC activators. Dialkyl 5-(hydroxymethyl)isophthalates are a novel group of synthetic C1 domain ligands that exhibit antiproliferative effect in HeLa cervical carcinoma cells. Here we selected two isophthalates, HMI-1a3 and HMI-1b11, and characterized their effects in the human neuroblastoma cell line SH-SY5Y. Both of the active isophthalates exhibited significant antiproliferative and differentiation-inducing effects. Since HMI-1b11 did not impair cell survival even at the highest concentration tested (20μM), and supported neurite growth and differentiation of SH-SY5Y cells, we focused on studying its downstream signaling cascades and effects on gene expression. Consistently, genome-wide gene expression microarray and gene set enrichment analysis indicated that HMI-1b11 (10μM) induced changes in genes mainly related to cell differentiation. In particular, further studies revealed that HMI-1b11 exposure induced up-regulation of GAP-43, a marker for neurite sprouting and neuronal differentiation. These effects were induced by a 7-min HMI-1b11 treatment and specifically depended on PKCα activation, since pretreatment with the selective inhibitor Gö6976 abolished the up-regulation of GAP-43 protein observed at 12h. In parallel, we found that a 7-min exposure to HMI-1b11 induced PKCα accumulation to the cytoskeleton, an effect that was again prevented by pretreatment with Gö6976. Despite similar binding affinities to PKC, the isophthalates had different effects on PKC-dependent ERK1/2 signaling: HMI-1a3-induced ERK1/2 phosphorylation was transient, while HMI-1b11 induced a rapid but prolonged ERK1/2 phosphorylation. Overall our data are in accordance with previous studies showing that activation of the PKCα and ERK1/2 pathways participate in regulating neuronal differentiation. Furthermore, since PKC has been classified as one of the cognitive kinases, and activation of PKC is considered a potential therapeutic strategy for the treatment of cognitive disorders, our findings suggest that HMI-1b11 represents a promising lead compound in research aimed to prevent or counteract memory impairment. Copyright © 2013 Elsevier Ltd. All rights reserved.

CAZymes Analysis Toolkit (CAT): web service for searching and analyzing carbohydrate-active enzymes in a newly sequenced organism using CAZy database.

PubMed

Park, Byung H; Karpinets, Tatiana V; Syed, Mustafa H; Leuze, Michael R; Uberbacher, Edward C

2010-12-01

The Carbohydrate-Active Enzyme (CAZy) database provides a rich set of manually annotated enzymes that degrade, modify, or create glycosidic bonds. Despite rich and invaluable information stored in the database, software tools utilizing this information for annotation of newly sequenced genomes by CAZy families are limited. We have employed two annotation approaches to fill the gap between manually curated high-quality protein sequences collected in the CAZy database and the growing number of other protein sequences produced by genome or metagenome sequencing projects. The first approach is based on a similarity search against the entire nonredundant sequences of the CAZy database. The second approach performs annotation using links or correspondences between the CAZy families and protein family domains. The links were discovered using the association rule learning algorithm applied to sequences from the CAZy database. The approaches complement each other and in combination achieved high specificity and sensitivity when cross-evaluated with the manually curated genomes of Clostridium thermocellum ATCC 27405 and Saccharophagus degradans 2-40. The capability of the proposed framework to predict the function of unknown protein domains and of hypothetical proteins in the genome of Neurospora crassa is demonstrated. The framework is implemented as a Web service, the CAZymes Analysis Toolkit, and is available at http://cricket.ornl.gov/cgi-bin/cat.cgi.

Gap junction- and hemichannel-independent actions of connexins.

PubMed

Jiang, Jean X; Gu, Sumin

2005-06-10

Connexins have been known to be the protein building blocks of gap junctions and mediate cell-cell communication. In contrast to the conventional dogma, recent evidence suggests that in addition to forming gap junction channels, connexins possess gap junction-independent functions. One important gap junction-independent function for connexins is to serve as the major functional component for hemichannels, the un-apposed halves of gap junctions. Hemichannels, as independent functional units, play roles that are different from that of gap junctions in the cell. The other functions of connexins appear to be gap junction- and hemichannel-independent. Published studies implicate the latter functions of connexins in cell growth, differentiation, tumorigenicity, injury, and apoptosis, although the mechanistic aspects of these actions remain largely unknown. In this review, gap junction- and hemichannel-independent functions of connexins are summarized, and the molecular mechanisms underlying these connexin functions are speculated and discussed.

[Study on diversity of protein between Houttuynia cordata plant].

PubMed

Zhang, Xi-li; He, Fu-yuan; Wang, Hai-qin; Yang, Yan-tao; Shi, Ji-lian; Liu, Wen-long; Li, Shun-xiang

2013-12-01

To reveal protein diversity between the same batch of fresh Houttuynia cordata in the same GAP base,and to lay the foundation construction for "node metabolic network". Three methods including the Ramagli improved Bradford law, SDS-PAGE gel electrophoresis method and double wavelength thin-layer scanning method were used to study the total protein content diversity, protein species diversity and various kinds of content variability. The molecular weight of 53 plant protein mostly concentrated in the range of 6.5-97.2 kDa, the species diversity was not obvious with main performance for banding color shades; The RSD of zero moment (AUCT), first moment (MCRTT) and second moment (VCRTT) in protein electrophoresis banding was 40.92%, 6.01% and 18.57%, respectively. There is rich diversity in different Houttuynia cordata plant in the same GAP base, which provides basis for the foundation of subsequent key protease search, "node metabolic network" construction, and study on the Chinese medicine quality stability.

Prenatal caffeine intake differently affects synaptic proteins during fetal brain development.

PubMed

Mioranzza, Sabrina; Nunes, Fernanda; Marques, Daniela M; Fioreze, Gabriela T; Rocha, Andréia S; Botton, Paulo Henrique S; Costa, Marcelo S; Porciúncula, Lisiane O

2014-08-01

Caffeine is the psychostimulant most consumed worldwide. However, little is known about its effects during fetal brain development. In this study, adult female Wistar rats received caffeine in drinking water (0.1, 0.3 and 1.0 g/L) during the active cycle in weekdays, two weeks before mating and throughout pregnancy. Cerebral cortex and hippocampus from embryonic stages 18 or 20 (E18 or E20, respectively) were collected for immunodetection of the following synaptic proteins: brain-derived neurotrophic factor (BDNF), TrkB receptor, Sonic Hedgehog (Shh), Growth Associated Protein 43 (GAP-43) and Synaptosomal-associated Protein 25 (SNAP-25). Besides, the estimation of NeuN-stained nuclei (mature neurons) and non-neuronal nuclei was verified in both brain regions and embryonic periods. Caffeine (1.0 g/L) decreased the body weight of embryos at E20. Cortical BDNF at E18 was decreased by caffeine (1.0 g/L), while it increased at E20, with no major effects on TrkB receptors. In the hippocampus, caffeine decreased TrkB receptor only at E18, with no effects on BDNF. Moderate and high doses of caffeine promoted an increase in Shh in both brain regions at E18, and in the hippocampus at E20. Caffeine (0.3g/L) decreased GAP-43 only in the hippocampus at E18. The NeuN-stained nuclei increased in the cortex at E20 by lower dose and in the hippocampus at E18 by moderate dose. Our data revealed that caffeine transitorily affect synaptic proteins during fetal brain development. The increased number of NeuN-stained nuclei by prenatal caffeine suggests a possible acceleration of the telencephalon maturation. Although some modifications in the synaptic proteins were transient, our data suggest that caffeine even in lower doses may alter the fetal brain development. Copyright © 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

Connexin-deficiency affects expression levels of glial glutamate transporters within the cerebrum.

PubMed

Unger, Tina; Bette, Stefanie; Zhang, Jiong; Theis, Martin; Engele, Jürgen

2012-01-06

The glial glutamate transporter subtypes, GLT-1/EAAT-2 and GLAST/EAAT-1 clear the bulk of extracellular glutamate and are severely dysregulated in various acute and chronic brain diseases. Despite the previous identification of several extracellular factors modulating glial glutamate transporter expression, our knowledge of the regulatory network controlling glial glutamate transport in health and disease still remains incomplete. In studies with cultured cortical astrocytes, we previously obtained evidence that glial glutamate transporter expression is also affected by gap junctions/connexins. To assess whether gap junctions would likewise control the in vivo expression of glial glutamate transporters, we have now assessed their expression levels in brains of conditional Cx43 knockout mice, total Cx30 knockouts, as well as Cx43/Cx30 double knockouts. We found that either knocking out Cx30, Cx43, or both increases GLT-1/EAAT-2 protein levels in the cerebral cortex to a similar extent. By contrast, GLAST/EAAT-1 protein levels maximally increased in cerebral cortices of Cx30/Cx43 double knockouts, implying that gap junctions differentially affect the expression of GLT-1/EAAT-2 and GLAST/EAAT-1. Quantitative PCR analysis further revealed that increases in glial glutamate transporter expression are brought about by transcriptional and translational/posttranslational processes. Moreover, GLT-1/EAAT-2- and GLAST/EAAT-1 protein levels remained unchanged in the hippocampi of Cx43/Cx30 double knockouts when compared to Cx43fl/fl controls, indicating brain region-specific effects of gap junctions on glial glutamate transport. Since astrocytic gap junction coupling is affected in various forms of brain injuries, our findings point to gap junctions/connexins as important regulators of glial glutamate turnover in the diseased cerebral cortex. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Toxicity of ZnO and TiO2 to Escherichia coli cells

PubMed Central

Leung, Yu Hang; Xu, Xiaoying; Ma, Angel P. Y.; Liu, Fangzhou; Ng, Alan M. C.; Shen, Zhiyong; Gethings, Lee A.; Guo, Mu Yao; Djurišić, Aleksandra B.; Lee, Patrick K. H.; Lee, Hung Kay; Chan, Wai Kin; Leung, Frederick C. C.

2016-01-01

We performed a comprehensive investigation of the toxicity of ZnO and TiO2 nanoparticles using Escherichia coli as a model organism. Both materials are wide band gap n-type semiconductors and they can interact with lipopolysaccharide molecules present in the outer membrane of E. coli, as well as produce reactive oxygen species (ROS) under UV illumination. Despite the similarities in their properties, the response of the bacteria to the two nanomaterials was fundamentally different. When the ROS generation is observed, the toxicity of nanomaterial is commonly attributed to oxidative stress and cell membrane damage caused by lipid peroxidation. However, we found that significant toxicity does not necessarily correlate with up-regulation of ROS-related proteins. TiO2 exhibited significant antibacterial activity, but the protein expression profile of bacteria exposed to TiO2 was different compared to H2O2 and the ROS-related proteins were not strongly expressed. On the other hand, ZnO exhibited lower antibacterial activity compared to TiO2, and the bacterial response involved up-regulating ROS-related proteins similar to the bacterial response to the exposure to H2O2. Reasons for the observed differences in toxicity and bacterial response to the two metal oxides are discussed. PMID:27731373

Satellite glial cells in the trigeminal ganglion as a determinant of orofacial neuropathic pain

PubMed Central

VIT, JEAN-PHILIPPE; JASMIN, LUC; BHARGAVA, ADITI; OHARA, PETER T.

2008-01-01

Satellite glial cells (SGCs) tightly envelop the perikarya of primary sensory neurons in peripheral ganglion and are identified by their morphology and the presence of proteins not found in ganglion neurons. These SGC-unique proteins include the inwardly rectifying K+ channel Kir4.1, the connexin-43 (Cx43) subunit of gap junctions, the purinergic receptor P2Y4 and soluble guanylate cyclase. We also present evidence that the small-conductance Ca2+-activated K+ channel SK3 is present only in SGCs and that SGCs divide following nerve injury. All the above proteins are involved, either directly or indirectly, in potassium ion (K+) buffering and, thus, can influence the level of neuronal excitability, which, in turn, has been associated with neuropathic pain conditions. We used in vivo RNA interference to reduce the expression of Cx43 (present only in SGCs) in the rat trigeminal ganglion and show that this results in the development of spontaneous pain behavior. The pain behavior is present only when Cx43 is reduced and returns to normal when Cx43 concentrations are restored. This finding shows that perturbation of a single SGC-specific protein is sufficient to induce pain responses and demonstrates the importance of PNS glial cell activity in the pathophysiology of neuropathic pain. PMID:18568096

CcpN Controls Central Carbon Fluxes in Bacillus subtilis▿ ‡

PubMed Central

Tännler, Simon; Fischer, Eliane; Le Coq, Dominique; Doan, Thierry; Jamet, Emmanuel; Sauer, Uwe; Aymerich, Stéphane

2008-01-01

The transcriptional regulator CcpN of Bacillus subtilis has been recently characterized as a repressor of two gluconeogenic genes, gapB and pckA, and of a small noncoding regulatory RNA, sr1, involved in arginine catabolism. Deletion of ccpN impairs growth on glucose and strongly alters the distribution of intracellular fluxes, rerouting the main glucose catabolism from glycolysis to the pentose phosphate (PP) pathway. Using transcriptome analysis, we show that during growth on glucose, gapB and pckA are the only protein-coding genes directly repressed by CcpN. By quantifying intracellular fluxes in deletion mutants, we demonstrate that derepression of pckA under glycolytic condition causes the growth defect observed in the ccpN mutant due to extensive futile cycling through the pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate kinase. Beyond ATP dissipation via this cycle, PckA activity causes a drain on tricarboxylic acid cycle intermediates, which we show to be the main reason for the reduced growth of a ccpN mutant. The high flux through the PP pathway in the ccpN mutant is modulated by the flux through the alternative glyceraldehyde-3-phosphate dehydrogenases, GapA and GapB. Strongly increased concentrations of intermediates in upper glycolysis indicate that GapB overexpression causes a metabolic jamming of this pathway and, consequently, increases the relative flux through the PP pathway. In contrast, derepression of sr1, the third known target of CcpN, plays only a marginal role in ccpN mutant phenotypes. PMID:18586936

Connexin hemichannels mediate glutathione transport and protect lens fiber cells from oxidative stress.

PubMed

Shi, Wen; Riquelme, Manuel A; Gu, Sumin; Jiang, Jean X

2018-03-21

Elevated oxidized stress contributes to lens cataracts, and gap junctions play important roles in maintaining lens transparency. As well as forming gap junctions, connexin (Cx) proteins also form hemichannels. Here, we report a new mechanism whereby hemichannels mediate transport of reductant glutathione into lens fiber cells and protect cells against oxidative stress. We found that Cx50 (also known as GJA8) hemichannels opened in response to H 2 O 2 in lens fiber cells but that transport through the channels was inhibited by two dominant-negative mutants in Cx50, Cx50P88S, which inhibits transport through both gap junctions and hemichannels, and Cx50H156N, which only inhibits transport through hemichannels and not gap junctions. Treatment with H 2 O 2 increased the number of fiber cells undergoing apoptosis, and this increase was augmented with dominant-negative mutants that disrupted both hemichannels formed from Cx46 (also known as GJA3) and Cx50, while Cx50E48K, which only impairs gap junctions, did not have such an effect. Moreover, hemichannels mediate uptake of glutathione, and this uptake protected lens fiber cells against oxidative stress, while hemichannels with impaired transport had less protective benefit from glutathione. Taken together, these results show that oxidative stress activates connexin hemichannels in the lens fiber cells and that hemichannels likely protect lens cell against oxidative damage through transporting extracellular reductants. © 2018. Published by The Company of Biologists Ltd.

The alpha2-adrenoreceptor agonist dexmedetomidine protects against lipopolysaccharide-induced apoptosis via inhibition of gap junctions in lung fibroblasts.

PubMed

Zhang, Yuan; Tan, Xiaoming; Xue, Lianfang

2018-01-01

The α2-adrenoceptor inducer dexmedetomidine protects against acute lung injury (ALI), but the mechanism of this effect is largely unknown. The present study investigated the effect of dexmedetomidine on apoptosis induced by lipopolysaccharide (LPS) and the relationship between this effect and gap junction intercellular communication in human lung fibroblast cell line. Flow cytometry was used to detect apoptosis induced by LPS. Parachute dye coupling assay was used to measure gap junction function, and western blot analysis was used to determine the expression levels of connexin43 (Cx43). The results revealed that exposure of human lung fibroblast cell line to LPS for 24 h increased the apoptosis, and pretreatment of dexmedetomidine and 18α-GA significantly reduced LPS-induced apoptosis. Dexmedetomidine exposure for 1 h inhibited gap junction function mainly via a decrease in Cx43 protein levels in human lung fibroblast cell line. These results demonstrated that the inhibition of gap junction intercellular communication by dexmedetomidine affected the LPS-induced apoptosis through inhibition of gap junction function by reducing Cx43 protein levels. The present study provides evidence of a novel mechanism underlying the effects of analgesics in counteracting ALI. Copyright © 2017 Elsevier Inc. All rights reserved.

Computational Models of Neuronal Biophysics and the Characterization of Potential Neuropharmacological Targets

PubMed Central

Ferrante, Michele; Blackwell, Kim T.; Migliore, Michele; Ascoli, Giorgio A.

2012-01-01

The identification and characterization of potential pharmacological targets in neurology and psychiatry is a fundamental problem at the intersection between medicinal chemistry and the neurosciences. Exciting new techniques in proteomics and genomics have fostered rapid progress, opening numerous questions as to the functional consequences of ligand binding at the systems level. Psycho- and neuro-active drugs typically work in nerve cells by affecting one or more aspects of electrophysiological activity. Thus, an integrated understanding of neuropharmacological agents requires bridging the gap between their molecular mechanisms and the biophysical determinants of neuronal function. Computational neuroscience and bioinformatics can play a major role in this functional connection. Robust quantitative models exist describing all major active membrane properties under endogenous and exogenous chemical control. These include voltage-dependent ionic channels (sodium, potassium, calcium, etc.), synaptic receptor channels (e.g. glutamatergic, GABAergic, cholinergic), and G protein coupled signaling pathways (protein kinases, phosphatases, and other enzymatic cascades). This brief review of neuromolecular medicine from the computational perspective provides compelling examples of how simulations can elucidate, explain, and predict the effect of chemical agonists, antagonists, and modulators in the nervous system. PMID:18855673

[Regulatory mechanisms in focal cerebral ischemia. New possibilities in neuroprotective therapy].

PubMed

Nagy, Zoltán; Simon, László; Bori, Zoltán

2002-03-20

Permanent or temporary disruption of cerebral blood flow rapidly depletes brain regions of their limited energy reserves (glycogen, glucose, oxygen, ATP) leading to an energy crisis. Tissue damage occurs due to the energy crisis. The central part of the damage, the ischaemic "core" region is surrounded by zones of the shell-like penumbra. Necrotic, as well as apoptotic cell death could be identified in the penumbra. Going away from the ischaemic core different neurochemical processes are occurring by space and time. "Immediate early response" genes (c-fos, fos-B, c-Jun, krox 20, 24) are activated, heatshock proteins (hsp 70, 72, HSF, HSE, HIF), cytokines (TNF-alpha, IL-1 beta), inflammatory factors (COX), adhesion and glial factors (ICAM-1, ELAM-1, P-selectin), vasoactive factors (IL-6, -10, PAF), reactive oxigen radicals and connected factors (O2, OH, NO, NOS, SOD) are produced within minutes and hours. Cell deaths, necrosis and apoptosis due to the activation of calpains, caspases and nucleases occur in days. In parallel, growth factors and plasticity proteins (BDNF, NGF, TGF-beta, VEGF, PDGF, GAP-43) are activated as a basis of functional rehabilitation.

Activity-dependent expression of ELAV/Hu RBPs and neuronal mRNAs in seizure and cocaine brain.

PubMed

Tiruchinapalli, Dhanrajan M; Caron, Marc G; Keene, Jack D

2008-12-01

Growing evidence indicates that both seizure (glutamate) and cocaine (dopamine) treatment modulate synaptic plasticity within the mesolimbic region of the CNS. Activation of glutamatergic neurons depends on the localized translation of neuronal mRNA products involved in modulating synaptic plasticity. In this study, we demonstrate the dendritic localization of HuR and HuD RNA-binding proteins (RBPs) and their association with neuronal mRNAs following these two paradigms of seizure and cocaine treatment. Both the ubiquitously expressed HuR and neuronal HuD RBPs were detected in different regions as well as within dendrites of the brain and in dissociated neurons. Quantitative analysis revealed an increase in HuR, HuD and p-glycogen synthase kinase 3beta (GSK3beta) protein levels as well as neuronal mRNAs encoding Homer, CaMKIIalpha, vascular early response gene, GAP-43, neuritin, and neuroligin protein products following either seizure or cocaine treatment. Inhibition of the Akt/GSK3beta signaling pathway by acute or chronic LiCl treatment revealed changes in HuR, HuD, pGSK3beta, p-Akt, and beta-catenin protein levels. In addition, a genetically engineered hyperdopaminergic mouse model (dopamine transporter knockout) revealed decreased expression of HuR protein levels, but no significant change was observed in HuD or fragile-X mental retardation protein RBPs. Finally, our data suggest that HuR and HuD RBPs potentially interact directly with neuronal mRNAs important for differentiation and synaptic plasticity.

A Dynamic Response Regulator Protein Modulates G-Protein–Dependent Polarity in the Bacterium Myxococcus xanthus

PubMed Central

Zhang, Yong; Guzzo, Mathilde; Ducret, Adrien; Li, Yue-Zhong; Mignot, Tâm

2012-01-01

Migrating cells employ sophisticated signal transduction systems to respond to their environment and polarize towards attractant sources. Bacterial cells also regulate their polarity dynamically to reverse their direction of movement. In Myxococcus xanthus, a GTP-bound Ras-like G-protein, MglA, activates the motility machineries at the leading cell pole. Reversals are provoked by pole-to-pole switching of MglA, which is under the control of a chemosensory-like signal transduction cascade (Frz). It was previously known that the asymmetric localization of MglA at one cell pole is regulated by MglB, a GTPase Activating Protein (GAP). In this process, MglB specifically localizes at the opposite lagging cell pole and blocks MglA localization at that pole. However, how MglA is targeted to the leading pole and how Frz activity switches the localizations of MglA and MglB synchronously remained unknown. Here, we show that MglA requires RomR, a previously known response regulator protein, to localize to the leading cell pole efficiently. Specifically, RomR-MglA and RomR-MglB complexes are formed and act complementarily to establish the polarity axis, segregating MglA and MglB to opposite cell poles. Finally, we present evidence that Frz signaling may regulate MglA localization through RomR, suggesting that RomR constitutes a link between the Frz-signaling and MglAB polarity modules. Thus, in Myxococcus xanthus, a response regulator protein governs the localization of a small G-protein, adding further insight to the polarization mechanism and suggesting that motility regulation evolved by recruiting and combining existing signaling modules of diverse origins. PMID:22916026

Msx1 and Msx2 are functional interacting partners of T-box factors in the regulation of Connexin43.

PubMed

Boogerd, Kees-Jan; Wong, L Y Elaine; Christoffels, Vincent M; Klarenbeek, Meinke; Ruijter, Jan M; Moorman, Antoon F M; Barnett, Phil

2008-06-01

T-box factors Tbx2 and Tbx3 play key roles in the development of the cardiac conduction system, atrioventricular canal, and outflow tract of the heart. They regulate the gap-junction-encoding gene Connexin43 (Cx43) and other genes critical for heart development and function. Discovering protein partners of Tbx2 and Tbx3 will shed light on the mechanisms by which these factors regulate these gene programs. Employing an yeast 2-hybrid screen and subsequent in vitro pull-down experiments we demonstrate that muscle segment homeobox genes Msx1 and Msx2 are able to bind the cardiac T-box proteins Tbx2, Tbx3, and Tbx5. This interaction, as that of the related Nkx2.5 protein, is supported by the T-box and homeodomain alone. Overlapping spatiotemporal expression patterns of Msx1 and Msx2 together with the T-box genes during cardiac development in mouse and chicken underscore the biological significance of this interaction. We demonstrate that Msx proteins together with Tbx2 and Tbx3 suppress Cx43 promoter activity and down regulate Cx43 gene activity in a rat heart-derived cell line. Using chromatin immunoprecipitation analysis we demonstrate that Msx1 can bind the Cx43 promoter at a conserved binding site located in close proximity to a previously defined T-box binding site, and that the activity of Msx proteins on this promoter appears dependent in the presence of Tbx3. Msx1 and Msx2 can function in concert with the T-box proteins to suppress Cx43 and other working myocardial genes.

A history of gap junction structure: hexagonal arrays to atomic resolution.

PubMed

Grosely, Rosslyn; Sorgen, Paul L

2013-02-01

Gap junctions are specialized membrane structures that provide an intercellular pathway for the propagation and/or amplification of signaling cascades responsible for impulse propagation, cell growth, and development. Prior to the identification of the proteins that comprise gap junctions, elucidation of channel structure began with initial observations of a hexagonal nexus connecting apposed cellular membranes. Concomitant with technological advancements spanning over 50 years, atomic resolution structures are now available detailing channel architecture and the cytoplasmic domains that have helped to define mechanisms governing the regulation of gap junctions. Highlighted in this review are the seminal structural studies that have led to our current understanding of gap junction biology.

Gap Junction Protein Connexin 43 Serves as a Negative Marker for a Stem Cell-Containing Population of Human Limbal Epithelial Cells

PubMed Central

Chen, Zhuo; Evans, W. Howard; Pflugfelder, Stephen C.; Li, De-Quan

2010-01-01

This study evaluated whether the gap junction protein connexin (Cx) 43 could serve as a negative cell surface marker for human corneal epithelial stem cells. Cx43 expression was evaluated in corneo-limbal tissue and primary limbal epithelial cultures. Immunofluorescent staining and laser scanning confocal microscopy showed that Cx43 was strongly expressed in the corneal and limbal suprabasal epithelial cells, but the basal cells of the limbal epithelium were negative. Cx43 antibody stained mainly large cells but not small cells in primary limbal epithelial cultures. As determined by semiquantitative reverse transcription polymerase chain reaction (PCR) and real-time PCR, Cx43 mRNA was more abundant in the corneal than limbal epithelia, and it was expressed in higher levels in mature limbal epithelial cultures. Using GAP11, a rabbit polyclonal antibody against the Cx32 extracellular loop 2 (151–187), a sequence that is highly homologous in Cx43, the Cx43dim and Cx43bright cells were selected from primary limbal epithelial cultures by fluorescence-activated cell sorting and were evaluated for stem cell properties. These Cx43dim and Cx43bright cells were confirmed by their expression levels of Cx43 protein and mRNA. The Cx43dim cells were found to contain higher percentages of slow-cycling bromodeoxyuridine (BrdU)-label retaining cells and the cells that were positive for stem cell-associated markers p63, ABCG2, and integrin β1 and negative for differentiation markers K3 and involucrin. The Cx43dim cells possessed a greater proliferative potential than Cx43bright cells and nonfractionated cells as evaluated by BrdU incorporation, colony-forming efficiency, and growth capacity. Our findings suggest that human limbal basal cells do not express connexin 43, which could serve as a negative cell surface marker for the stem cell-containing population of human limbal epithelial cells. PMID:16424398

Nanoscale On-Silico Electron Transport via Ferritins.

PubMed

Bera, Sudipta; Kolay, Jayeeta; Banerjee, Siddhartha; Mukhopadhyay, Rupa

2017-02-28

Silicon is a solid-state semiconducting material that has long been recognized as a technologically useful one, especially in electronics industry. However, its application in the next-generation metalloprotein-based electronics approaches has been limited. In this work, the applicability of silicon as a solid support for anchoring the iron-storage protein ferritin, which has a semiconducting iron nanocore, and probing electron transport via the ferritin molecules trapped between silicon substrate and a conductive scanning probe has been investigated. Ferritin protein is an attractive bioelectronic material because its size (X-ray crystallographic diameter ∼12 nm) should allow it to fit well in the larger tunnel gaps (>5 nm), fabrication of which is relatively more established, than the smaller ones. The electron transport events occurring through the ferritin molecules that are covalently anchored onto the MPTMS-modified silicon surface could be detected at the molecular level by current-sensing atomic force spectroscopy (CSAFS). Importantly, the distinct electronic signatures of the metal types (i.e., Fe, Mn, Ni, and Au) within the ferritin nanocore could be distinguished from each other using the transport band gap analyses. The CSAFS measurements on holoferritin, apoferritin, and the metal core reconstituted ferritins reveal that some of these ferritins behave like n-type semiconductors, while the others behave as p-type semiconductors. The band gaps for the different ferritins are found to be within 0.8 to 2.6 eV, a range that is valid for the standard semiconductor technology (e.g., diodes based on p-n junction). The present work indicates effective on-silico integration of the ferritin protein, as it remains functionally viable after silicon binding and its electron transport activities can be detected. Potential use of the ferritin-silicon nanohybrids may therefore be envisaged in applications other than bioelectronics, too, as ferritin is a versatile nanocore-containing biomaterial (for storage/transport of metals and drugs) and silicon can be a versatile nanoscale solid support (for its biocompatible nature).

Redox-mediated regulation of connexin proteins; focus on nitric oxide.

PubMed

García, Isaac E; Sánchez, Helmuth A; Martínez, Agustín D; Retamal, Mauricio A

2018-01-01

Connexins are membrane proteins that form hemichannels and gap junction channels at the plasma membrane. Through these channels connexins participate in autocrine and paracrine intercellular communication. Connexin-based channels are tightly regulated by membrane potential, phosphorylation, pH, redox potential, and divalent cations, among others, and the imbalance of this regulation have been linked to many acquired and genetic diseases. Concerning the redox potential regulation, the nitric oxide (NO) has been described as a modulator of the hemichannels and gap junction channels properties. However, how NO regulates these channels is not well understood. In this mini-review, we summarize the current knowledge about the effects of redox potential focused in NO on the trafficking, formation and functional properties of hemichannels and gap junction channels. Copyright © 2017 Elsevier B.V. All rights reserved.

Assessing the impact of a medical librarian on identification of valid and actionable practice gaps for a continuing medical education committee.

PubMed

Bartkowiak, Barbara A; Safford, Lindsey A; Stratman, Erik J

2014-01-01

Identifying educational needs related to professional practice gaps can be a complex process for continuing medical education (CME) committees and for physicians who submit activity applications. Medical librarians possess unique skills that may be useful for identifying practice gaps relevant to CME committees. We assessed this assumption by assessing a medical librarian's contributions to practice gap identification for the Marshfield Clinic's CME Committee. We reviewed all locally relevant, locally actionable practice gaps identified annually by various stakeholders and presented to our CME Committee from 2010 to 2013. Total numbers of practice gaps identified, total categorized as actionable, and numbers of subsequent activities resulting from these gaps were calculated for each year. Medical librarian totals were compared to those of other CME committee stakeholders to determine the relative contribution. The medical librarian identified unique, actionable published practice gaps that directly contributed to CME activity planning. For each study year, contributions by the medical librarian grew, from 0 of 27 actionable gaps validated by CME Committee in 2010 to 49 of 108 (45.4%) in 2013. With the librarian's assistance, the number of valid practice gaps submitted between 2010 and 2013 by stakeholders climbed from 23 for 155 activities (14.8%) to 133 for 157 activities (84.7%). Medical librarians can provide a valuable service to CME committees by identifying valid professional practice gaps that inform decisions about educational activities aimed at improving clinical practice. Medical librarians bring into deliberations unique information, including national health policy priorities, practice gaps found in the literature, and point-of-care search engine statistics. © 2014 The Alliance for Continuing Education in the Health Professions, the Society for Academic Continuing Medical Education, and the Council on Continuing Medical Education, Association for Hospital Medical Education.

Hippocampal synapsin I, growth-associated protein-43, and microtubule-associated protein-2 immunoreactivity in learned helplessness rats and antidepressant-treated rats.

PubMed

Iwata, M; Shirayama, Y; Ishida, H; Kawahara, R

2006-09-01

Learned helplessness rats are thought to be an animal model of depression. To study the role of synapse plasticity in depression, we examined the effects of learned helplessness and antidepressant treatments on synapsin I (a marker of presynaptic terminals), growth-associated protein-43 (GAP-43; a marker of growth cones), and microtubule-associated protein-2 (MAP-2; a marker of dendrites) in the hippocampus by immunolabeling. (1) Learned helplessness rats showed significant increases in the expression of synapsin I two days after the attainment of learned helplessness, and significant decreases in the protein expression eight days after the achievement of learned helplessness. Subchronic treatment of naïve rats with imipramine or fluvoxamine significantly decreased the expression of synapsin I. (2) Learned helplessness increased the expression of GAP-43 two days and eight days after learned helplessness training. Subchronic treatment of naïve rats with fluvoxamine but not imipramine showed a tendency to decrease the expression of synapsin I. (3) Learned helplessness rats showed increased expression of MAP-2 eight days after the attainment of learned helplessness. Naïve rats subchronically treated with imipramine showed a tendency toward increased expression of MAP-2, but those treated with fluvoxamine did not. These results indicate that the neuroplasticity-related proteins synapsin I, GAP-43, and MAP-2 may play a role in the pathophysiology of depression and the mechanisms of antidepressants.

Characterization of the Structure and Intermolecular Interactions between the Connexin 32 Carboxyl-terminal Domain and the Protein Partners Synapse-associated Protein 97 and Calmodulin*

PubMed Central

Stauch, Kelly; Kieken, Fabien; Sorgen, Paul

2012-01-01

In Schwann cells, connexin 32 (Cx32) can oligomerize to form intracellular gap junction channels facilitating a shorter pathway for metabolite diffusion across the layers of the myelin sheath. The mechanisms of Cx32 intracellular channel regulation have not been clearly defined. However, Ca2+, pH, and the phosphorylation state can regulate Cx32 gap junction channels, in addition to the direct interaction of protein partners with the carboxyl-terminal (CT) domain. In this study, we used different biophysical methods to determine the structure and characterize the interaction of the Cx32CT domain with the protein partners synapse-associated protein 97 (SAP97) and calmodulin (CaM). Our results revealed that the Cx32CT is an intrinsically disordered protein that becomes α-helical upon binding CaM. We identified the GUK domain as the minimal SAP97 region necessary for the Cx32CT interaction. The Cx32CT residues affected by the binding of CaM and the SAP97 GUK domain were determined as well as the dissociation constants for these interactions. We characterized three Cx32CT Charcot-Marie-Tooth disease mutants (R219H, R230C, and F235C) and identified that whereas they all formed functional channels, they all showed reduced binding affinity for SAP97 and CaM. Additionally, we report that in RT4-D6P2T rat schwannoma cells, Cx32 is differentially phosphorylated and exists in a complex with SAP97 and CaM. Our studies support the importance of protein-protein interactions in the regulation of Cx32 gap junction channels and myelin homeostasis. PMID:22718765

Microtubule-assisted altered trafficking of astrocytic gap junction protein connexin 43 is associated with depletion of connexin 47 during mouse hepatitis virus infection.

PubMed

Basu, Rahul; Bose, Abhishek; Thomas, Deepthi; Das Sarma, Jayasri

2017-09-08

Gap junctions (GJs) are important for maintenance of CNS homeostasis. GJ proteins, connexin 43 (Cx43) and connexin 47 (Cx47), play a crucial role in production and maintenance of CNS myelin. Cx43 is mainly expressed by astrocytes in the CNS and forms gap junction intercellular communications between astrocytes-astrocytes (Cx43-Cx43) and between astrocytes-oligodendrocytes (Cx43-Cx47). Mutations of these connexin (Cx) proteins cause dysmyelinating diseases in humans. Previously, it has been shown that Cx43 localization and expression is altered due to mouse hepatitis virus (MHV)-A59 infection both in vivo and in vitro ; however, its mechanism and association with loss of myelin protein was not elaborated. Thus, we explored potential mechanisms by which MHV-A59 infection alters Cx43 localization and examined the effects of viral infection on Cx47 expression and its association with loss of the myelin marker proteolipid protein. Immunofluorescence and total internal reflection fluorescence microscopy confirmed that MHV-A59 used microtubules (MTs) as a conduit to reach the cell surface and restricted MT-mediated Cx43 delivery to the cell membrane. Co-immunoprecipitation experiments demonstrated that Cx43-β-tubulin molecular interaction was depleted due to protein-protein interaction between viral particles and MTs. During acute MHV-A59 infection, oligodendrocytic Cx47, which is mainly stabilized by Cx43 in vivo , was down-regulated, and its characteristic staining remained disrupted even at chronic phase. The loss of Cx47 was associated with loss of proteolipid protein at the chronic stage of MHV-A59 infection. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Rsr1 Focuses Cdc42 Activity at Hyphal Tips and Promotes Maintenance of Hyphal Development in Candida albicans

PubMed Central

Pulver, Rebecca; Heisel, Timothy; Gonia, Sara; Robins, Robert; Norton, Jennifer; Haynes, Paula

2013-01-01

The extremely elongated morphology of fungal hyphae is dependent on the cell's ability to assemble and maintain polarized growth machinery over multiple cell cycles. The different morphologies of the fungus Candida albicans make it an excellent model organism in which to study the spatiotemporal requirements for constitutive polarized growth and the generation of different cell shapes. In C. albicans, deletion of the landmark protein Rsr1 causes defects in morphogenesis that are not predicted from study of the orthologous protein in the related yeast Saccharomyces cerevisiae, thus suggesting that Rsr1 has expanded functions during polarized growth in C. albicans. Here, we show that Rsr1 activity localizes to hyphal tips by the differential localization of the Rsr1 GTPase-activating protein (GAP), Bud2, and guanine nucleotide exchange factor (GEF), Bud5. In addition, we find that Rsr1 is needed to maintain the focused localization of hyphal polarity structures and proteins, including Bem1, a marker of the active GTP-bound form of the Rho GTPase, Cdc42. Further, our results indicate that tip-localized Cdc42 clusters are associated with the cell's ability to express a hyphal transcriptional program and that the ability to generate a focused Cdc42 cluster in early hyphae (germ tubes) is needed to maintain hyphal morphogenesis over time. We propose that in C. albicans, Rsr1 “fine-tunes” the distribution of Cdc42 activity and that self-organizing (Rsr1-independent) mechanisms of polarized growth are not sufficient to generate narrow cell shapes or to provide feedback to the transcriptional program during hyphal morphogenesis. PMID:23223038

The receptor protein tyrosine phosphatase (RPTP){beta}/{zeta} is expressed in different subtypes of human breast cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perez-Pinera, Pablo; Garcia-Suarez, Olivia; Instituto Universitario de Oncologia del Principado de Asturias, Oviedo

2007-10-12

Increasing evidence suggests mutations in human breast cancer cells that induce inappropriate expression of the 18-kDa cytokine pleiotrophin (PTN, Ptn) initiate progression of breast cancers to a more malignant phenotype. Pleiotrophin signals through inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP){beta}/{zeta}, leading to increased tyrosine phosphorylation of different substrate proteins of RPTP{beta}/{zeta}, including {beta}-catenin, {beta}-adducin, Fyn, GIT1/Cat-1, and P190RhoGAP. PTN signaling thus has wide impact on different important cellular systems. Recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTP{beta}/{zeta} signaling pathway; this discovery potentially is very important, since constitutive ALK activity of nucleophosmin (NPM)-ALK fusionmore » protein is causative of anaplastic large cell lymphomas, and, activated ALK is found in other malignant cancers. Recently ALK was identified in each of 63 human breast cancers from 22 subjects. We now demonstrate that RPTP{beta}/{zeta} is expressed in each of these same 63 human breast cancers that previously were found to express ALK and in 10 additional samples of human breast cancer. RPTP{beta}/{zeta} furthermore was localized not only in its normal association with the cell membrane but also scattered in cytoplasm and in nuclei in different breast cancer cells and, in the case of infiltrating ductal carcinomas, the distribution of RPTP{beta}/{zeta} changes as the breast cancer become more malignant. The data suggest that the PTN/RPTP{beta}/{zeta} signaling pathway may be constitutively activated and potentially function to constitutively activate ALK in human breast cancer.« less

The Structural Basis of Oncogenic Mutations G12, G13 and Q61 in Small GTPase K-Ras4B

NASA Astrophysics Data System (ADS)

Lu, Shaoyong; Jang, Hyunbum; Nussinov, Ruth; Zhang, Jian

2016-02-01

Ras mediates cell proliferation, survival and differentiation. Mutations in K-Ras4B are predominant at residues G12, G13 and Q61. Even though all impair GAP-assisted GTP → GDP hydrolysis, the mutation frequencies of K-Ras4B in human cancers vary. Here we aim to figure out their mechanisms and differential oncogenicity. In total, we performed 6.4 μs molecular dynamics simulations on the wild-type K-Ras4B (K-Ras4BWT-GTP/GDP) catalytic domain, the K-Ras4BWT-GTP-GAP complex, and the mutants (K-Ras4BG12C/G12D/G12V-GTP/GDP, K-Ras4BG13D-GTP/GDP, K-Ras4BQ61H-GTP/GDP) and their complexes with GAP. In addition, we simulated ‘exchanged’ nucleotide states. These comprehensive simulations reveal that in solution K-Ras4BWT-GTP exists in two, active and inactive, conformations. Oncogenic mutations differentially elicit an inactive-to-active conformational transition in K-Ras4B-GTP; in K-Ras4BG12C/G12D-GDP they expose the bound nucleotide which facilitates the GDP-to-GTP exchange. These mechanisms may help elucidate the differential mutational statistics in K-Ras4B-driven cancers. Exchanged nucleotide simulations reveal that the conformational transition is more accessible in the GTP-to-GDP than in the GDP-to-GTP exchange. Importantly, GAP not only donates its R789 arginine finger, but stabilizes the catalytically-competent conformation and pre-organizes catalytic residue Q61; mutations disturb the R789/Q61 organization, impairing GAP-mediated GTP hydrolysis. Together, our simulations help provide a mechanistic explanation of key mutational events in one of the most oncogenic proteins in cancer.

Immunogenicity of amino acids 1-150 of Streptococcus GapC displayed on the surface of Escherichia coli.

PubMed

Song, Baifen; Yang, Xijing; Sun, Hunan; Yu, Liquan; Ma, Jinzhu; Wu, Zhijun; Cui, Yudong

2017-04-01

Streptococcus is one of the main pathogens that cause bovine mastitis. They includes into S.agalactiae, S.dysgalactiae, and S.uberis. The GapC protein is a virulence factor that is expressed on the surface of Streptococcus species. GapC is highly antigenic and immunization with GapC confers cross-protection against all three species. Our previous data showed that amino acids 1-150 of GapC (GapC 1-150 ) of S. dysgalactiae conferred similar immunoprotection compared to full-length GapC. Thus, the present study aimed to construct a recombinant Escherichia coli XL1-Blue strain that displayed GapC 1-150 on its surface, and to investigate the immunogenicity of the surface-localized GapC 1-150 . To do so, the ompA gene of the E. coli XL1-Blue strain was replaced with the lpp'-ompA-gapC1 1-150 or lpp'-ompA genes by λ Red recombination, the former of which fused GapC 1-150 to an Lpp lipoprotein signal peptide and amino acids 1-159 of OmpA; the recombinant strains were named XL1-Blue/LOG76 and XL1-Blue/LO11, respectively. GapC 1-150 was confirmed to localize to the surface of the XL1-Blue/LOG76 strain by an indirect enzyme-linked immunosorbent assay (ELISA), a fluorescence-activated cell sorter analysis, and laser-scanning confocal microscopy. Then, ICR mice were immunized intramuscularly with the XL1-Blue/LOG76 or XL1-Blue/LO11 strains, or recombinant GapC 1-150 . The sera of the immunized mice were collected and the anti-GapC 1-150 antibody levels were detected by ELISA. Lymphocytes secreting interleukin (IL)-4 and interferon-γ were detected by an enzyme-linked ImmunoSpot assay, as was the level of IL-17A level in the supernatant of cultured splenic lymphocytes. The mice immunized with the XL1-Blue/LOG76 strain or GapC 1-150 exhibited better cellular and humoral immunity. Lastly, the immunized mice were challenged with S. uberis, S. dysgalactiae, and S. agalactiae strains, and mice that were immunized with the XL1-Blue/LOG76 strain were better protected than those that were immunized with the XL1-Blue/LO11 strain. These results indicate that it is feasible to display GapC 1-150 on the E. coli surface as a vaccine against Streptococcus species. Copyright © 2017. Published by Elsevier Ltd.

A Novel Heat Shock Element (HSE) in Entamoeba histolytica that Regulates the Transcriptional Activation of the EhPgp5 Gene in the Presence of Emetine Drug.

PubMed

Nieto, Alma; Pérez Ishiwara, David G; Orozco, Esther; Sánchez Monroy, Virginia; Gómez García, Consuelo

2017-01-01

Transcriptional regulation of the multidrug resistance EhPgp5 gene in Entamoeba histolytica is induced by emetine stress. EhPgp5 overexpression alters the chloride-dependent currents that cause trophozoite swelling, diminishing induced programmed cell death (PCD) susceptibility. In contrast, antisense inhibition of P-glycoprotein (PGP) expression produces synchronous death of trophozoites and the enhancement of the biochemical and morphological characteristics of PCD induced by G418. Transcriptional gene regulation analysis identified a 59 bp region at position -170 to -111 bp promoter as putative emetine response elements (EREs). However, insights into transcription factors controlling EhPgp5 gene transcription are missing; to fill this knowledge gap, we used deletion studies and transient CAT activity assays. Our findings suggested an activating motif (-151 to -136 bp) that corresponds to a heat shock element (HSE). Gel-shift assays, UV-crosslinking, binding protein purification, and western blotting assays revealed proteins of 94, 66, 62, and 51 kDa binding to the EhPgp5 HSE that could be heat shock-like transcription factors that regulate the transcriptional activation of the EhPgp5 gene in the presence of emetine drug.

A multiplexed single-cell CRISPR screening platform enables systematic dissection of the unfolded protein response

PubMed Central

Adamson, Britt; Norman, Thomas M.; Jost, Marco; Cho, Min Y.; Nuñez, James K.; Chen, Yuwen; Villalta, Jacqueline E.; Gilbert, Luke A.; Horlbeck, Max A.; Hein, Marco Y.; Pak, Ryan A.; Gray, Andrew N.; Gross, Carol A.; Dixit, Atray; Parnas, Oren; Regev, Aviv; Weissman, Jonathan S.

2016-01-01

SUMMARY Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ~100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses. PMID:27984733

Connexin-dependent gap junction enhancement is involved in the synergistic effect of sorafenib and all-trans retinoic acid on HCC growth inhibition.

PubMed

Yang, Yan; Qin, Shu-Kui; Wu, Qiong; Wang, Zi-Shu; Zheng, Rong-Sheng; Tong, Xu-Hui; Liu, Hao; Tao, Liang; He, Xian-Di

2014-02-01

Increasing gap junction activity in tumor cells provides a target by which to enhance antineoplastic therapies. Previously, several naturally occurring agents, including all-trans retinoic acid (ATRA) have been demonstrated to increase gap junctional intercellular communication (GJIC) in a number of types of cancer cells. In the present study, we investigated in vitro whether ATRA modulates the response of human hepatocellular carcinoma (HCC) cells to sorafenib, the only proven oral drug for advanced HCC, and the underlying mechanisms. HepG2 and SMMC-7721 cells were treated with sorafenib and/or ATRA, and cell proliferation and apoptosis were analyzed; the role of GJIC was also explored. We found that ATRA, at non-toxic concentrations, enhanced sorafenib-induced growth inhibition in both HCC cell lines, and this effect was abolished by two GJIC inhibitors, 18-α-GA and oleamide. Whereas lower concentrations of sorafenib (5 µM) or ATRA (0.1 or 10 µM) alone modestly induced GJIC activity, the combination of sorafenib plus ATRA resulted in a strong enhancement of GJIC. However, the action paradigm differed in the HepG2 and SMMC-7721 cells, with the dominant effect of GJIC dependent on the cell-specific connexin increase in protein amounts and relocalization. RT-PCR assay further revealed a transcriptional modification of the key structural connexin in the two cell lines. Thus, a connexin-dependent gap junction enhancement may play a central role in ATRA plus sorafenib synergy in inhibiting HCC cell growth. Since both agents are available for human use, the combination treatment represents a future profitable strategy for the treatment of advanced HCC.

Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix.

PubMed

Ambrosi, Cinzia; Walker, Amy E; Depriest, Adam D; Cone, Angela C; Lu, Connie; Badger, John; Skerrett, I Martha; Sosinsky, Gina E

2013-01-01

Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure.

The Carboxyl Tail of Connexin32 Regulates Gap Junction Assembly in Human Prostate and Pancreatic Cancer Cells*

PubMed Central

Katoch, Parul; Mitra, Shalini; Ray, Anuttoma; Kelsey, Linda; Roberts, Brett J.; Wahl, James K.; Johnson, Keith R.; Mehta, Parmender P.

2015-01-01

Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size. PMID:25548281

Evidence that ganglion cells react to retinal detachment.

PubMed

Coblentz, Francie E; Radeke, Monte J; Lewis, Geoffrey P; Fisher, Steven K

2003-03-01

Growth associated protein 43 (GAP 43) is involved in synapse formation and it is expressed in the retina in a very specific pattern. Although GAP 43 is downregulated at the time of synapse formation, it can be re-expressed following injury such as axotomy or ischemia. Because of this we sought to characterize the expression of GAP 43 after retinal detachment (RD). Immunoblot, immunocytochemical and quantitative polymerase chain reaction (QPCR) techniques were used to assess the level of GAP 43 expression after experimental RD. GAP 43 was localized to three sublaminae of the inner plexiform layer of the normal retina. GAP 43 became upregulated in a subset of retinal ganglion cells following at least 7 days of RD. By immunoblot GAP 43 could be detected by 3 days. QPCR shows the upregulation of GAP 43 message by 6hr of detachment. To further characterize changes in ganglion cells, we used an antibody to neurofilament 70 and 200kDa (NF) proteins. Anti-NF labels horizontal cells, ganglion cell dendrites in the inner plexiform layer, and ganglion cell axons (fasicles) in the normal retina. Following detachment it is upregulated in horizontal cells and ganglion cells. When detached retina was double labelled with anti-GAP 43 and anti-NF, some cells were labelled with both markers, while others labelled with only one. We have previously shown that second order neurons respond to detachment; here we show that third order neurons are responding as well. Cellular remodelling of this type in response to detachment may explain the slow recovery of vision that often occurs after reattachment, or those changes that are often assumed to be permanent.

Mechanisms of gap gene expression canalization in the Drosophila blastoderm.

PubMed

Gursky, Vitaly V; Panok, Lena; Myasnikova, Ekaterina M; Manu; Samsonova, Maria G; Reinitz, John; Samsonov, Alexander M

2011-01-01

Extensive variation in early gap gene expression in the Drosophila blastoderm is reduced over time because of gap gene cross regulation. This phenomenon is a manifestation of canalization, the ability of an organism to produce a consistent phenotype despite variations in genotype or environment. The canalization of gap gene expression can be understood as arising from the actions of attractors in the gap gene dynamical system. In order to better understand the processes of developmental robustness and canalization in the early Drosophila embryo, we investigated the dynamical effects of varying spatial profiles of Bicoid protein concentration on the formation of the expression border of the gap gene hunchback. At several positions on the anterior-posterior axis of the embryo, we analyzed attractors and their basins of attraction in a dynamical model describing expression of four gap genes with the Bicoid concentration profile accounted as a given input in the model equations. This model was tested against a family of Bicoid gradients obtained from individual embryos. These gradients were normalized by two independent methods, which are based on distinct biological hypotheses and provide different magnitudes for Bicoid spatial variability. We showed how the border formation is dictated by the biological initial conditions (the concentration gradient of maternal Hunchback protein) being attracted to specific attracting sets in a local vicinity of the border. Different types of these attracting sets (point attractors or one dimensional attracting manifolds) define several possible mechanisms of border formation. The hunchback border formation is associated with intersection of the spatial gradient of the maternal Hunchback protein and a boundary between the attraction basins of two different point attractors. We demonstrated how the positional variability for hunchback is related to the corresponding variability of the basin boundaries. The observed reduction in variability of the hunchback gene expression can be accounted for by specific geometrical properties of the basin boundaries. We clarified the mechanisms of gap gene expression canalization in early Drosophila embryos. These mechanisms were specified in the case of hunchback in well defined terms of the dynamical system theory.

Broad gap junction blocker carbenoxolone disrupts uterine preparation for embryo implantation in mice.

PubMed

Diao, Honglu; Xiao, Shuo; Howerth, Elizabeth W; Zhao, Fei; Li, Rong; Ard, Mary B; Ye, Xiaoqin

2013-08-01

Gap junctions have an important role in cell-to-cell communication, a process obviously required for embryo implantation. Uterine luminal epithelium (LE) is the first contact for an implanting embryo and is critical for the establishment of uterine receptivity. Microarray analysis of the LE from peri-implantation mouse uterus showed low-level expression of 19 gap junction proteins in preimplantation LE and upregulation of gap junction protein, beta 2 (GJB2, connexin 26, Cx26) in postimplantation LE. Time course study using in situ hybridization and immunofluorescence revealed upregulation of GJB2 in the LE surrounding the implantation site before decidualization. Similar dynamic expression of GJB2 was observed in the LE of artificially decidualized mice but not pseudopregnant mice. To determine the potential function of uterine gap junctions in embryo implantation, carbenoxolone (CBX), a broad gap junction blocker, was injected i.p. (100 mg/kg) or via local uterine fat pad (10 mg/kg) into pregnant mice on Gestation Day 3 at 1800 h, a few hours before embryo attachment to the LE. These CBX treatments disrupted embryo implantation, suggesting local effects of CBX in the uterus. However, i.p. injection of glycyrrhizic acid (100 mg/kg), which shares similar structure and multiple properties with CBX but is ineffective in blocking gap junctions, did not affect embryo implantation. Carbenoxolone also inhibited oil-induced artificial decidualization, concomitant with suppressed molecular changes and ultrastructural transformations associated with uterine preparation for embryo implantation, underscoring the adverse effect of CBX on uterine preparation for embryo implantation. These data demonstrate that uterine gap junctions are important for embryo implantation.

Measuring impact of JAMA Dermatology Practice Gaps section on training in US dermatology residency programs.

PubMed

Britton, Kristina M; Stratman, Erik J

2013-07-01

JAMA Dermatology Practice Gaps commentaries are intended to aid in the interpretation of the literature to make it more practical and applicable to daily patient care. Practice Gaps commentaries have had an impact on physician clinical practice and dermatology residency curricula. To assess the impact of JAMA Dermatology Practice Gaps commentaries on dermatology residency training programs in the United States, including journal club discussions and local quality improvement activities. A web-based questionnaire of 17 questions was sent via e-mail to US dermatology residency program directors (PDs) in February 2012. Program director report of incorporating Practice Gaps themes and discussions into resident journal club activities, clinical practice, quality improvement activities, or research projects in the residency programs, as a result of a Practice Gaps commentary. Of the 114 surveys distributed to US dermatology residency PDs, 48 were completed (42% response rate). Sixty percent of PDs reported familiarity with the Practice Gaps section of JAMA Dermatology, and 56% discuss these commentaries during resident journal club activities. Quality improvement and research projects have been initiated as a result of Practice Gaps commentaries. Practice Gaps commentaries are discussed during most dermatology residency journal club activities. Practice Gaps have had an impact on physician practice and dermatology residency curricula and can serve as a tool for enhanced continuing medical education and quality improvement initiatives.

Bombyx mori cyclin-dependent kinase inhibitor is involved in regulation of the silkworm cell cycle.

PubMed

Tang, X-F; Zhou, X-L; Zhang, Q; Chen, P; Lu, C; Pan, M-H

2018-06-01

Cyclin-dependent kinase inhibitors (CKIs) are negative regulators of the cell cycle. They can bind to cyclin-dependent kinase (CDK)-cyclin complexes and inhibit CDK activities. We identified a single homologous gene of the CDK interacting protein/kinase inhibitory protein (Cip/Kip) family, BmCKI, in the silkworm, Bombyx mori. The gene transcribes two splice variants: a 654-bp-long BmCKI-L (the longer splice variant) encoding a protein with 217 amino acids and a 579-bp-long BmCKI-S (the shorter splice variant) encoding a protein with 192 amino acids. BmCKI-L and BmCKI-S contain the Cip/Kip family conserved cyclin-binding domain and the CDK-binding domain. They are localized in the nucleus and have an unconventional bipartite nuclear localization signal at amino acid residues 181-210. Overexpression of BmCKI-L or BmCKI-S affected cell cycle progression; the cell cycle was arrested in the first gap phase of cell cycle (G1). RNA interference of BmCKI-L or BmCKI-S led to cells accumulating in the second gap phase and the mitotic phase of cell cycle (G2/M). Both BmCKI-L and BmCKI-S are involved in cell cycle regulation and probably have similar effects. The transgenic silkworm with BmCKI-L overexpression (BmCKI-L-OE), exhibited embryonic lethal, larva developmental retardation and lethal phenotypes. These results suggest that BmCKI-L might regulate the growth and development of silkworm. These findings clarify the function of CKIs and increase our understanding of cell cycle regulation in the silkworm. © 2018 The Royal Entomological Society.

Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.

PubMed

Reith, A D; Ellis, C; Lyman, S D; Anderson, D M; Williams, D E; Bernstein, A; Pawson, T

1991-09-01

Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c-kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c-kit gene products with cytoplasmic signalling proteins, using transient c-kit expression assays in COS cells. In addition to the previously identified c-kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an in-frame insertion, Gly-Asn-Asn-Lys, within the extracellular domain, was detected in murine mast cell cultures and mid-gestation placenta. Both Kit+ and KitA+ isoforms showed increased autophosphorylation and enhanced association with phosphatidylinositol (PI) 3' kinase and PLC gamma 1, when stimulated with recombinant soluble Steel factor. No association or increase in phosphorylation of GAP and two GAP-associated proteins, p62 and p190, was observed. The two isoforms had distinct activities in the absence of exogenous soluble Steel factor; Kit+, but not KitA+, showed constitutive tyrosine phosphorylation that was accompanied by a low constitutive level of association with PI-3' kinase and PLC gamma 1. Introduction of the point substitutions associated with W37 (Glu582----Lys) or W41 (Val831----Met) mutant alleles into c-kit expression constructs abolished (W37) or reduced (W41) the Steel factor-induced association of the Kit receptor with signalling proteins in a manner proportional to the overall severity of the corresponding W mutant phenotype. These data suggest a diversity of normal Kit signalling pathways and indicate that W mutant phenotypes result from primary defects in the Kit receptor that affect its interaction with cytoplasmic signalling proteins.

An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layer segregation

NASA Technical Reports Server (NTRS)

Wikramanayake, Athula H.; Hong, Melanie; Lee, Patricia N.; Pang, Kevin; Byrum, Christine A.; Bince, Joanna M.; Xu, Ronghui; Martindale, Mark Q.

2003-01-01

The human oncogene beta-catenin is a bifunctional protein with critical roles in both cell adhesion and transcriptional regulation in the Wnt pathway. Wnt/beta-catenin signalling has been implicated in developmental processes as diverse as elaboration of embryonic polarity, formation of germ layers, neural patterning, spindle orientation and gap junction communication, but the ancestral function of beta-catenin remains unclear. In many animal embryos, activation of beta-catenin signalling occurs in blastomeres that mark the site of gastrulation and endomesoderm formation, raising the possibility that asymmetric activation of beta-catenin signalling specified embryonic polarity and segregated germ layers in the common ancestor of bilaterally symmetrical animals. To test whether nuclear translocation of beta-catenin is involved in axial identity and/or germ layer formation in 'pre-bilaterians', we examined the in vivo distribution, stability and function of beta-catenin protein in embryos of the sea anemone Nematostella vectensis (Cnidaria, Anthozoa). Here we show that N. vectensis beta-catenin is differentially stabilized along the oral-aboral axis, translocated into nuclei in cells at the site of gastrulation and used to specify entoderm, indicating an evolutionarily ancient role for this protein in early pattern formation.

The F-BAR domains from srGAP1, srGAP2 and srGAP3 regulate membrane deformation differently

PubMed Central

Coutinho-Budd, Jaeda; Ghukasyan, Vladimir; Zylka, Mark J.; Polleux, Franck

2012-01-01

Summary Coordination of membrane deformation and cytoskeletal dynamics lies at the heart of many biological processes critical for cell polarity, motility and morphogenesis. We have recently shown that Slit-Robo GTPase-activating protein 2 (srGAP2) regulates neuronal morphogenesis through the ability of its F-BAR domain to regulate membrane deformation and induce filopodia formation. Here, we demonstrate that the F-BAR domains of two closely related family members, srGAP1 and srGAP3 [designated F-BAR(1) and F-BAR(3), respectively] display significantly different membrane deformation properties in non-neuronal COS7 cells and in cortical neurons. F-BAR(3) induces filopodia in both cell types, though less potently than F-BAR(2), whereas F-BAR(1) prevents filopodia formation in cortical neurons and reduces plasma membrane dynamics. These three F-BAR domains can heterodimerize, and they act synergistically towards filopodia induction in COS7 cells. As measured by fluorescence recovery after photobleaching, F-BAR(2) displays faster molecular dynamics than F-BAR(3) and F-BAR(1) at the plasma membrane, which correlates well with its increased potency to induce filopodia. We also show that the molecular dynamic properties of F-BAR(2) at the membrane are partially dependent on F-Actin. Interestingly, acute phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] depletion in cells does not interfere with plasma membrane localization of F-BAR(2), which is compatible with our result showing that F-BAR(2) binds to a broad range of negatively-charged phospholipids present at the plasma membrane, including phosphatidylserine (PtdSer). Overall, our results provide novel insights into the functional diversity of the membrane deformation properties of this subclass of F-BAR-domains required for cell morphogenesis. PMID:22467852

Improving cardiac gap junction communication as a new antiarrhythmic mechanism: the action of antiarrhythmic peptides.

PubMed

Dhein, Stefan; Hagen, Anja; Jozwiak, Joanna; Dietze, Anna; Garbade, Jens; Barten, Markus; Kostelka, Martin; Mohr, Friedrich-Wilhelm

2010-03-01

Co-ordinated electrical activation of the heart is maintained by intercellular coupling of cardiomyocytes via gap junctional channels located in the intercalated disks. These channels consist of two hexameric hemichannels, docked to each other, provided by either of the adjacent cells. Thus, a complete gap junction channel is made from 12 protein subunits, the connexins. While 21 isoforms of connexins are presently known, cardiomyocytes typically are coupled by Cx43 (most abundant), Cx40 or Cx45. Some years ago, antiarrhythmic peptides were discovered and synthesised, which were shown to increase macroscopic gap junction conductance (electrical coupling) and enhance dye transfer (metabolic coupling). The lead substance of these peptides is AAP10 (H-Gly-Ala-Gly-Hyp-Pro-Tyr-CONH(2)), a peptide with a horseshoe-like spatial structure as became evident from two-dimensional nuclear magnetic resonance studies. A stable D: -amino-acid derivative of AAP10, rotigaptide, as well as a non-peptide analogue, gap-134, has been developed in recent years. Antiarrhythmic peptides act on Cx43 and Cx45 gap junctions but not on Cx40 channels. AAP10 has been shown to enhance intercellular communication in rat, rabbit and human cardiomyocytes. Antiarrhythmic peptides are effective against ventricular tachyarrhythmias, such as late ischaemic (type IB) ventricular fibrillation, CaCl(2) or aconitine-induced arrhythmia. Interestingly, the effect of antiarrhythmic peptides is higher in partially uncoupled cells and was shown to be related to maintained Cx43 phosphorylation, while arrhythmogenic conditions like ischaemia result in Cx43 dephosphorylation and intercellular decoupling. It is still a matter of debate whether these drugs also act against atrial fibrillation. The present review outlines the development of this group of peptides and derivatives, their mode of action and molecular mechanisms, and discusses their possible therapeutic potential.

Gap junction blockade induces apoptosis in human endometrial stromal cells.

PubMed

Yu, Jie; Berga, Sarah L; Zou, Wei; Sun, He-Ying; Johnston-MacAnanny, Erika; Yalcinkaya, Tamer; Sidell, Neil; Bagchi, Indrani C; Bagchi, Milan K; Taylor, Robert N

2014-07-01

One of the most dynamic adult human tissues is the endometrium. Through coordinated, cyclical proliferation, differentiation, leukocyte recruitment, apoptosis, and desquamation, the uterine lining is expanded and shed monthly, unless pregnancy is established. Errors in these steps potentially cause endometrial dysfunction, abnormal uterine bleeding, failed embryonic implantation, infertility, or endometrial carcinoma. Our prior studies showed that gap junctions comprised of Gap junction alpha-1 (GJA1) protein, also known as connexin 43 (CX43), subunits are critical to endometrial stromal cell differentiation. The current studies were undertaken to explore the mechanism of endometrial dysfunction when gap junction intercellular communication (GJIC) is disrupted. Gap junction blockade by two distinct GJIC inhibitors, 18α-glycyrrhetinic acid (AGA) and octanol (OcOH), suppressed proliferation and induced apoptosis in endometrial stromal cells, as manifested by reduced biomarkers of cell viability, increased TUNEL staining, caspase-3 activation, sub-G1 chromosomal DNA complement, as well as shortened telomere length. Unexpectedly, we also observed that the chemical inhibitors blocked CX43 gene expression. Moreover, when endometrial stromal cells were induced to undergo hormonal decidualization, following a 7-day exposure to 10 nM 17β-estradiol + 100 nM progesterone + 0.5 mM dibutyryl cAMP, characteristic epithelioid changes in cell shape and secretion of prolactin were blunted in the presence of AGA or OcOH, recapitulating effects of RNA interference of CX43. Our findings indicate that endometrial stromal cell proliferation and maintenance of decidualized endometrial function are GJIC-dependent, and that disruption of gap junctions induces endometrial stromal cell apoptosis. These observations may have important implications for several common clinical endometrial pathologies. © 2014 Wiley Periodicals, Inc.

Regulation of osteoclastogenesis by gap junction communication.

PubMed

Matemba, Stephen F; Lie, Anita; Ransjö, Maria

2006-10-01

Receptor activator of NF-kappaB ligand (RANKL) is crucial in osteoclastogenesis but signaling events involved in osteoclast differentiation are far from complete and other signals may play a role in osteoclastogenesis. A more direct pathway for cellular crosstalk is provided by gap junction intercellular channel, which allows adjacent cells to exchange second messengers, ions, and cellular metabolites. Here we have investigated the role of gap junction communication in osteoclastogenesis in mouse bone marrow cultures. Immunoreactive sites for the gap junction protein connexin 43 (Cx43) were detected in the marrow stromal cells and in mature osteoclasts. Carbenoxolone (CBX) functionally blocked gap junction communication as demonstrated by a scrape loading Lucifer Yellow dye transfer technique. CBX caused a dose-dependent inhibition (significant > or = 90 microM) of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed in 7- to 8-day marrow cultures stimulated by parathyroid hormone (PTH; 10 nM) or forskolin (FSK; 1 microM). Furthermore, CBX (100 microM) significantly inhibited prostaglandin E2 (PGE2; 10 microM) and 1,25(OH)2-vitamin D3 stimulated osteoclast differentiation in the mouse bone marrow cultures. Consequently, quantitative real-time polymerase chain reaction (PCR) analysis demonstrated that CBX downregulated the expression of osteoclast phenotypic markers, but without having any significant effects on RANK, RANKL, and osteoprotegerin (OPG) mRNA expression. However, the results demonstrated that CBX significantly inhibits RANKL-stimulated (100 ng/ml) osteoclastogenesis in the mouse bone marrow cultures. Taken together, our results suggests that gap junctional diffusion of messenger molecules interacts with signaling pathways downstream RANKL in osteoclast differentiation. Further studies are required to define the precise mechanisms and molecular targets involved. Copyright 2006 Wiley-Liss, Inc.

Growth associated protein (GAP-43): cloning and the development of a sensitive ELISA for neurological disorders.

PubMed

Gnanapavan, Sharmilee; Yousaf, Nasim; Heywood, Wendy; Grant, Donna; Mills, Kevin; Chernajovsky, Yuti; Keir, Geoff; Giovannoni, Gavin

2014-11-15

GAP-43 has been studied in the rodent and mammalian brain and shown to be present specifically in areas undergoing axonal elongation and synapse formation. GAP-43 was cloned using the baculovirus expression system and purified. A sandwich ELISA was developed using the recombinant GAP-43 as standard and validated. CSF GAP-43 levels were analysed in benign intracranial hypertension, movement disorders, multiple sclerosis, neuropathy, CNS infections, motor neuron disease, and headache (neurological controls). GAP-43 levels were low in all disorders analysed (in particular motor neuron disease; p=0.001, and movement disorders and multiple sclerosis; p<0.0001) compared to controls, aside from CNS infections. GAP-43 is preferentially reduced in the CSF of neurological disorders associated with neurodegeneration. Copyright © 2014. Published by Elsevier B.V.

Free-energy landscape of ion-channel voltage-sensor–domain activation

PubMed Central

Delemotte, Lucie; Kasimova, Marina A.; Klein, Michael L.; Tarek, Mounir; Carnevale, Vincenzo

2015-01-01

Voltage sensor domains (VSDs) are membrane-bound protein modules that confer voltage sensitivity to membrane proteins. VSDs sense changes in the transmembrane voltage and convert the electrical signal into a conformational change called activation. Activation involves a reorganization of the membrane protein charges that is detected experimentally as transient currents. These so-called gating currents have been investigated extensively within the theoretical framework of so-called discrete-state Markov models (DMMs), whereby activation is conceptualized as a series of transitions across a discrete set of states. Historically, the interpretation of DMM transition rates in terms of transition state theory has been instrumental in shaping our view of the activation process, whose free-energy profile is currently envisioned as composed of a few local minima separated by steep barriers. Here we use atomistic level modeling and well-tempered metadynamics to calculate the configurational free energy along a single transition from first principles. We show that this transition is intrinsically multidimensional and described by a rough free-energy landscape. Remarkably, a coarse-grained description of the system, based on the use of the gating charge as reaction coordinate, reveals a smooth profile with a single barrier, consistent with phenomenological models. Our results bridge the gap between microscopic and macroscopic descriptions of activation dynamics and show that choosing the gating charge as reaction coordinate masks the topological complexity of the network of microstates participating in the transition. Importantly, full characterization of the latter is a prerequisite to rationalize modulation of this process by lipids, toxins, drugs, and genetic mutations. PMID:25535341

Free-energy landscape of ion-channel voltage-sensor-domain activation.

PubMed

Delemotte, Lucie; Kasimova, Marina A; Klein, Michael L; Tarek, Mounir; Carnevale, Vincenzo

2015-01-06

Voltage sensor domains (VSDs) are membrane-bound protein modules that confer voltage sensitivity to membrane proteins. VSDs sense changes in the transmembrane voltage and convert the electrical signal into a conformational change called activation. Activation involves a reorganization of the membrane protein charges that is detected experimentally as transient currents. These so-called gating currents have been investigated extensively within the theoretical framework of so-called discrete-state Markov models (DMMs), whereby activation is conceptualized as a series of transitions across a discrete set of states. Historically, the interpretation of DMM transition rates in terms of transition state theory has been instrumental in shaping our view of the activation process, whose free-energy profile is currently envisioned as composed of a few local minima separated by steep barriers. Here we use atomistic level modeling and well-tempered metadynamics to calculate the configurational free energy along a single transition from first principles. We show that this transition is intrinsically multidimensional and described by a rough free-energy landscape. Remarkably, a coarse-grained description of the system, based on the use of the gating charge as reaction coordinate, reveals a smooth profile with a single barrier, consistent with phenomenological models. Our results bridge the gap between microscopic and macroscopic descriptions of activation dynamics and show that choosing the gating charge as reaction coordinate masks the topological complexity of the network of microstates participating in the transition. Importantly, full characterization of the latter is a prerequisite to rationalize modulation of this process by lipids, toxins, drugs, and genetic mutations.

Engineering protein self-assembling in protein-based nanomedicines for drug delivery and gene therapy.

PubMed

Ferrer-Miralles, Neus; Rodríguez-Carmona, Escarlata; Corchero, José Luis; García-Fruitós, Elena; Vázquez, Esther; Villaverde, Antonio

2015-06-01

Lack of targeting and improper biodistribution are major flaws in current drug-based therapies that prevent reaching high local concentrations of the therapeutic agent. Such weaknesses impose the administration of high drug doses, resulting in undesired side effects, limited efficacy and enhanced production costs. Currently, missing nanosized containers, functionalized for specific cell targeting will be then highly convenient for the controlled delivery of both conventional and innovative drugs. In an attempt to fill this gap, health-focused nanotechnologies have put under screening a growing spectrum of materials as potential components of nanocages, whose properties can be tuned during fabrication. However, most of these materials pose severe biocompatibility concerns. We review in this study how proteins, the most versatile functional macromolecules, can be conveniently exploited and adapted by conventional genetic engineering as efficient building blocks of fully compatible nanoparticles for drug delivery and how selected biological activities can be recruited to mimic viral behavior during infection. Although engineering of protein self-assembling is still excluded from fully rational approaches, the exploitation of protein nano-assemblies occurring in nature and the direct manipulation of protein-protein contacts in bioinspired constructs open intriguing possibilities for further development. These methodologies empower the construction of new and potent vehicles that offer promise as true artificial viruses for efficient and safe nanomedical applications.

A Homogeneous Polysaccharide from Fructus Schisandra chinensis (Turz.) Baill Induces Mitochondrial Apoptosis through the Hsp90/AKT Signalling Pathway in HepG2 Cells.

PubMed

Chen, Yonglin; Shi, Songshan; Wang, Huijun; Li, Ning; Su, Juan; Chou, Guixin; Wang, Shunchun

2016-06-28

According to the potential anti-hepatoma therapeutic effect of Schisandra chinensis polysaccharides presented in previous studies, a bioactive constituent, homogeneous Schisandra chinensis polysaccharide-0-1 (SCP-0-1), molecular weight (MW) circa 69.980 kDa, was isolated and purified. We assessed the efficacy of SCP-0-1 against human hepatocellular liver carcinoma (HepG2) cells to investigate the effects of its antitumour activity and molecular mechanisms. Anticancer activity was evaluated using microscopy, 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide (MTT) assay, Hoechst 33258 staining, acridine orange (AO) staining, flow cytometry (FCM), and cell-cycle analysis. SCP-0-1 inhibited the HepG2 cells' growth via inducing apoptosis and second gap/mitosis (G2/M) arrest dose-dependently, with a half maximal inhibitory concentration (IC50) value of 479.63 µg/mL. Western blotting of key proteins revealed the apoptotic and autophagic potential of SCP-0-1. Besides, SCP-0-1 upregulated Bcl-2 Associated X Protein (Bax) and downregulated B-cell leukemia/lymphoma 2 (Bcl-2) in the HepG2 cells. The expression of caspase-3, -8, and -9; poly (ADP-ribose) polymerase (PARP); cytochrome c (Cyt C); tumor protein 53 (p53); survivin; sequestosome 1 (p62); microtubule-associated protein 1 light chain-3B (LC3B); mitogen-activated protein kinase p38 (p38); extracellular regulated protein kinases (ERK); c-Jun N-terminal kinase (JNK); protein kinase B (AKT); and heat shock protein 90 (Hsp90) were evaluated using Western blotting. Our findings demonstrate a novel mechanism through which SCP-0-1 exerts its antiproliferative activity and induces mitochondrial apoptosis rather than autophagy. The induction of mitochondrial apoptosis was attributed to the inhibition of the Hsp90/AKT signalling pathway in an extracellular signal-regulated kinase-independent manner. The results also provide initial evidence on a molecular basis that SCP-0-1 can be used as an anti-hepatocellular carcinoma therapeutic agent in the future.

Stabilization, Rolling, and Addition of Other Extracellular Matrix Proteins to Collagen Hydrogels Improve Regeneration in Chitosan Guides for Long Peripheral Nerve Gaps in Rats.

PubMed

Gonzalez-Perez, Francisco; Cobianchi, Stefano; Heimann, Claudia; Phillips, James B; Udina, Esther; Navarro, Xavier

2017-03-01

Autograft is still the gold standard technique for the repair of long peripheral nerve injuries. The addition of biologically active scaffolds into the lumen of conduits to mimic the endoneurium of peripheral nerves may increase the final outcome of artificial nerve devices. Furthermore, the control of the orientation of the collagen fibers may provide some longitudinal guidance architecture providing a higher level of mesoscale tissue structure. To evaluate the regenerative capabilities of chitosan conduits enriched with extracellular matrix-based scaffolds to bridge a critical gap of 15 mm in the rat sciatic nerve. The right sciatic nerve of female Wistar Hannover rats was repaired with chitosan tubes functionalized with extracellular matrix-based scaffolds fully hydrated or stabilized and rolled to bridge a 15 mm nerve gap. Recovery was evaluated by means of electrophysiology and algesimetry tests and histological analysis 4 months after injury. Stabilized constructs enhanced the success of regeneration compared with fully hydrated scaffolds. Moreover, fibronectin-enriched scaffolds increased muscle reinnervation and number of myelinated fibers compared with laminin-enriched constructs. A mixed combination of collagen and fibronectin may be a promising internal filler for neural conduits for the repair of peripheral nerve injuries, and their stabilization may increase the quality of regeneration over long gaps. Copyright © 2017 by the Congress of Neurological Surgeons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rivedal, Edgar; Leithe, Edward

Benzene is used at large volumes in many different human activities. Hematotoxicity and cancer-causation as a result of benzene exposure was recognized many years ago, but the mechanisms involved remain unclear. Aberrant regulation of gap junction intercellular communication (GJIC) has been linked to both cancer induction and interference with normal hematopoietic development. We have previously suggested that inhibition of GJIC may play a role in benzene toxicity since benzene metabolites were found to block GJIC, the ring-opened trans,trans-muconaldehyde (MUC) being the most potent metabolite. In the present work we have studied the molecular mechanisms underlying the MUC-induced inhibition of gapmore » junctional communication. We show that MUC induces cross-linking of the gap junction protein connexin43 and that this is likely to be responsible for the induced inhibition of GJIC, as well as the loss of connexin43 observed in Western blots. We also show that glutaraldehyde possesses similar effects as MUC, and we compare the effects to that of formaldehyde. The fact that glutaraldehyde and formaldehyde have been associated with induction of leukemia as well as disturbance of hematopoiesis, strengthens the possible link between the effect of MUC on gap junctions, and the toxic effects of benzene.« less

Methoxychlor and Vinclozolin Induce Rapid Changes in Intercellular and Intracellular Signaling in Liver Progenitor Cells.

PubMed

Babica, Pavel; Zurabian, Rimma; Kumar, Esha R; Chopra, Rajus; Mianecki, Maxwell J; Park, Joon-Suk; Jaša, Libor; Trosko, James E; Upham, Brad L

2016-09-01

Methoxychlor (MXC) and vinclozolin (VIN) are well-recognized endocrine disrupting chemicals known to alter epigenetic regulations and transgenerational inheritance; however, non-endocrine disruption endpoints are also important. Thus, we determined the effects of MXC and VIN on the dysregulation of gap junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells. Both chemicals induced a rapid dysregulation of GJIC at non-cytotoxic doses, with 30 min EC50 values for GJIC inhibition being 10 µM for MXC and 126 µM for VIN. MXC inhibited GJIC for at least 24 h, while VIN effects were transient and GJIC recovered after 4 h. VIN induced rapid hyperphosphorylation and internalization of gap junction protein connexin43, and both chemicals also activated MAPK ERK1/2 and p38. Effects on GJIC were not prevented by MEK1/2 inhibitor, but by an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), resveratrol, and in the case of VIN, also, by a p38 inhibitor. Estrogen (ER) and androgen receptor (AR) modulators (estradiol, ICI 182,780, HPTE, testosterone, flutamide, VIN M2) did not attenuate MXC or VIN effects on GJIC. Our data also indicate that the effects were elicited by the parental compounds of MXC and VIN. Our study provides new evidence that MXC and VIN dysregulate GJIC via mechanisms involving rapid activation of PC-PLC occurring independently of ER- or AR-dependent genomic signaling. Such alterations of rapid intercellular and intracellular signaling events involved in regulations of gene expression, tissue development, function and homeostasis, could also contribute to transgenerational epigenetic effects of endocrine disruptors. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Comparative effects of torasemide and furosemide on gap junction proteins and cardiac fibrosis in a rat model of dilated cardiomyopathy.

PubMed

Watanabe, Kenichi; Sreedhar, Remya; Thandavarayan, Rajarajan A; Karuppagounder, Vengadeshprabhu; Giridharan, Vijayasree V; Antony, Shanish; Harima, Meilei; Nakamura, Masahiko; Suzuki, Kenji; Suzuki, Hiroshi; Sone, Hirohito; Arumugam, Somasundaram

2017-03-01

Cardiac fibrosis is the major hallmark of adverse cardiac remodeling in chronic heart failure (CHF) and its therapeutic targeting might help against cardiac dysfunction during chronic conditions. Diuretic agents are potentially useful in these cases, but their effects on the cardiac fibrosis pathogenesis are yet to be identified. This study was designed to identify and compare the effects of diuretic drugs torasemide and furosemide on cardiac fibrosis in a rat model of dilated cardiomyopathy induced by porcine cardiac myosin mediated experimental autoimmune myocarditis. Gap junction proteins, connexin-43 and N-cadherin, expressions were downregulated in the hearts of CHF rats, while torasemide treatment has upregulated their expression. Western blotting and immunohistochemical analysis for various cardiac fibrosis related proteins as well as histopathological studies have shown that both drugs have potential anti-fibrotic effects. Among them, torasemide has superior efficacy in offering protection against adverse cardiac remodeling in the selected rat model of dilated cardiomyopathy. In conclusion, torasemide treatment has potential anti-fibrotic effect in the hearts of CHF rats, possibly via improving the gap junction proteins expression and thereby improving the cell-cell interaction in the heart. © 2016 BioFactors, 43(2):187-194, 2017. © 2016 International Union of Biochemistry and Molecular Biology.

Insilico study of the A(2A)R-D (2)R kinetics and interfacial contact surface for heteromerization.

PubMed

Prakash, Amresh; Luthra, Pratibha Mehta

2012-10-01

G-protein-coupled receptors (GPCRs) are cell surface receptors. The dynamic property of receptor-receptor interactions in GPCRs modulates the kinetics of G-protein signaling and stability. In the present work, the structural and dynamic study of A(2A)R-D(2)R interactions was carried to acquire the understanding of the A(2A)R-D(2)R receptor activation and deactivation process, facilitating the design of novel drugs and therapeutic target for Parkinson's disease. The structure-based features (Alpha, Beta, SurfAlpha, and SurfBeta; GapIndex, Leakiness and Gap Volume) and slow mode model (ENM) facilitated the prediction of kinetics (K (off), K (on), and K (d)) of A(2A)R-D(2)R interactions. The results demonstrated the correlation coefficient 0.294 for K (d) and K (on) and the correlation coefficient 0.635 for K (d) and K (off), and indicated stable interfacial contacts in the formation of heterodimer. The coulombic interaction involving the C-terminal tails of the A(2A)R and intracellular loops (ICLs) of D(2)R led to the formation of interfacial contacts between A(2A)R-D(2)R. The properties of structural dynamics, ENM and KFC server-based hot-spot analysis illustrated the stoichiometry of A(2A)R-D(2)R contact interfaces as dimer. The propensity of amino acid residues involved in A(2A)R-D(2)R interaction revealed the presence of positively (R, H and K) and negatively (E and D) charged structural motif of TMs and ICL3 of A(2A)R and D(2)R at interface of dimer contact. Essentially, in silico structural and dynamic study of A(2A)R-D(2)R interactions will provide the basic understanding of the A(2A)R-D(2)R interfacial contact surface for activation and deactivation processes, and could be used as constructive model to recognize the protein-protein interactions in receptor assimilations.

Kinetic Measurements Reveal Enhanced Protein-Protein Interactions at Intercellular Junctions

PubMed Central

Shashikanth, Nitesh; Kisting, Meridith A.; Leckband, Deborah E.

2016-01-01

The binding properties of adhesion proteins are typically quantified from measurements with soluble fragments, under conditions that differ radically from the confined microenvironment of membrane bound proteins in adhesion zones. Using classical cadherin as a model adhesion protein, we tested the postulate that confinement within quasi two-dimensional intercellular gaps exposes weak protein interactions that are not detected in solution binding assays. Micropipette-based measurements of cadherin-mediated, cell-cell binding kinetics identified a unique kinetic signature that reflects both adhesive (trans) bonds between cadherins on opposing cells and lateral (cis) interactions between cadherins on the same cell. In solution, proposed lateral interactions were not detected, even at high cadherin concentrations. Mutations postulated to disrupt lateral cadherin association altered the kinetic signatures, but did not affect the adhesive (trans) binding affinity. Perturbed kinetics further coincided with altered cadherin distributions at junctions, wound healing dynamics, and paracellular permeability. Intercellular binding kinetics thus revealed cadherin interactions that occur within confined, intermembrane gaps but not in solution. Findings further demonstrate the impact of these revealed interactions on the organization and function of intercellular junctions. PMID:27009566

[Effect of Electroacupuncture on Expression of Cortical srGAP 1 and Cdc 42 in Rats with Focal Cerebral Infarction].

PubMed

Dai, En-ze; Long, Fei; Gong, Biao; Guo, Quan-hu; Wang, Ying; Zeng, Zhi-hua

2015-06-01

To observe the effect of electroacupuncture (EA) intervention on the neurological function and the expression change of Slit-Robo GTPase-activating protein-1 (srGAP 1) and cell division-cycle 42 (Cdc 42) in the cortex of rats with cerebral ischemic injury (CIRI) , so as to explore the mechanism of EA in the management of cerebral infarction. A total of 48 male Sprague Dawley (SD) rats were randomly and equally divided into control, model, non-acupoint EA and EA groups (n = 12/group). The CIRI model was established based on the modified Zea Longa method. EA intervention was applied for 30 min, once a day for 14 days. Modified neurologic severity scores (mNSS) were assessed on day 1,3,7 and 14 after mode- ling. Immunofluorescence assay was used to detect the immunoactivity and distribution of srGAP 1 and Cdc 42 in the cortical ischemic region. Western blot was employed to detect the expression of srGAP 1 and Cdc 42 in the affected cortex. The mNSS displayed that the neurological score in the EA group was significantly lower than that in the model group and non-acupoint EA group at the 7th d and 14th d (P<0. 01). Immunofluorescence results showed that cerebral srGAP 1 and Cdc 42 were ex- pressed mainly in the cytoplasm. The fluorescence intensity of srGAP 1 of the EA group was significantly lower than that of the model group and non-acupoint EA group(P<0. 01). Meanwhile the fluorescence intensity of Cdc 42 of the EA group was markedly higher than that in the model group and non-acupoint EA group(P<0. 01). Western blot assay indicated that the expression level of srGAP 1 in the model group was significantly higher than that of the control group( P<0. 01) ,and that of the EA group was much lower than those of the model group and non-acupoint EA group(P<0. 01). There was no significant difference of srGAP 1 expression levels between the non-acupoint EA group and the model group(P>0. 05). Additionally, the protein expression of Cdc 42 in the model group was slightly higher than that of the control group(P>0. 05), and that of the EA group was significantly higher than those of the model group and non-acupoint EA group(P<0. 01). There was no significant difference of Cdc 42 expression levels between the non-acupoint EA group and the model group(P>0. 05). Cerebral infarction induced increase of cerebral srGAP 1 and decrease of Cdc 42 can be reversed by acupoint EA intervention in CIRI rats, which may be responsible for its effect in improving impaired neurological function after cerebral infarction.

Ultrastructural localization of connexins (Cx36, Cx43, Cx45), glutamate receptors and aquaporin-4 in rodent olfactory mucosa, olfactory nerve and olfactory bulb

PubMed Central

RASH, JOHN E.; DAVIDSON, KIMBERLY G. V.; KAMASAWA, NAOMI; YASUMURA, THOMAS; KAMASAWA, MASAMI; ZHANG, CHUNBO; MICHAELS, ROBIN; RESTREPO, DIEGO; OTTERSEN, OLE P.; OLSON, CARL O.; NAGY, JAMES I.

2006-01-01

Odorant/receptor binding and initial olfactory information processing occurs in olfactory receptor neurons (ORNs) within the olfactory epithelium. Subsequent information coding involves high-frequency spike synchronization of paired mitral/tufted cell dendrites within olfactory bulb (OB) glomeruli via positive feedback between glutamate receptors and closely-associated gap junctions. With mRNA for connexins Cx36, Cx43 and Cx45 detected within ORN somata and Cx36 and Cx43 proteins reported in ORN somata and axons, abundant gap junctions were proposed to couple ORNs. We used freeze-fracture replica immunogold labeling (FRIL) and confocal immunofluorescence microscopy to examine Cx36, Cx43 and Cx45 protein in gap junctions in olfactory mucosa, olfactory nerve and OB in adult rats and mice and early postnatal rats. In olfactory mucosa, Cx43 was detected in gap junctions between virtually all intrinsic cell types except ORNs and basal cells; whereas Cx45 was restricted to gap junctions in sustentacular cells. ORN axons contained neither gap junctions nor any of the three connexins. In OB, Cx43 was detected in homologous gap junctions between almost all cell types except neurons and oligodendrocytes. Cx36 and, less abundantly, Cx45 were present in neuronal gap junctions, primarily at “mixed” glutamatergic/electrical synapses between presumptive mitral/tufted cell dendrites. Genomic analysis revealed multiple miRNA (micro interfering RNA) binding sequences in 3′-untranslated regions of Cx36, Cx43 and Cx45 genes, consistent with cell-type-specific post-transcriptional regulation of connexin synthesis. Our data confirm absence of gap junctions between ORNs, and support Cx36- and Cx45-containing gap junctions at glutamatergic mixed synapses between mitral/tufted cells as contributing to higher-order information coding within OB glomeruli. PMID:16841170

Relationship between connexin expression and gap-junction resistivity in human atrial myocardium.

PubMed

Dhillon, Paramdeep S; Chowdhury, Rasheda A; Patel, Pravina M; Jabr, Rita; Momin, Aziz U; Vecht, Joshua; Gray, Rosaire; Shipolini, Alex; Fry, Christopher H; Peters, Nicholas S

2014-04-01

The relative roles of the gap-junctional proteins connexin40 (Cx40) and connexin43 (Cx43) in determining human atrial myocardial resistivity is unknown. In addressing the hypothesis that changing relative expression of Cx40 and Cx43 underlies an increase in human atrial myocardial resistivity with age, this relationship was investigated by direct ex vivo measurement of gap-junctional resistivity and quantitative connexin immunoblotting and immunohistochemistry. Oil-gap impedance measurements were performed to determine resistivity of the intracellular pathway (Ri), which correlated with total Cx40 quantification by Western blotting (rs=0.64, P<0.01, n=20). Specific gap-junctional resistivity (Rj) correlated not only with Western immunoquantification of Cx40 (rs=0.63, P=0.01, n=20), but also more specifically, with the Cx40 fraction localized to the intercalated disks on immunohistochemical quantification (rs=0.66, P=0.02, n=12). Although Cx43 expression showed no correlation with resistivity values, the proportional expression of the 2 connexins, (Cx40/[Cx40+Cx43]) correlated with Ri and Rj (rs=0.58, P<0.01 for Ri and rs=0.51, P=0.02 for Rj). Advancing age was associated with a rise in Ri (rs=0.77, P<0.0001), Rj (rs=0.65, P<0.001, n=23), Cx40 quantity (rs=0.54, P=0.01, n=20), and Cx40 gap-junction protein per unit area of en face disk (rs=0.61, P=0.02, n=12). Cx40 is associated with human right atrial gap-junctional resistivity such that increased total, gap-junctional, and proportional Cx40 expression increases gap-junctional resistivity. Accordingly, advancing age is associated with an increase in Cx40 expression and a corresponding increase in gap-junctional resistivity. These findings are the first to demonstrate this relationship and a mechanistic explanation for changing atrial conduction and age-related arrhythmic tendency.

Characterization of carbonic anhydrase XIII in the erythrocytes of the Burmese python, Python molurus bivittatus.

PubMed

Esbaugh, A J; Secor, S M; Grosell, M

2015-09-01

Carbonic anhydrase (CA) is one of the most abundant proteins found in vertebrate erythrocytes with the majority of species expressing a low activity CA I and high activity CA II. However, several phylogenetic gaps remain in our understanding of the expansion of cytoplasmic CA in vertebrate erythrocytes. In particular, very little is known about isoforms from reptiles. The current study sought to characterize the erythrocyte isoforms from two squamate species, Python molurus and Nerodia rhombifer, which was combined with information from recent genome projects to address this important phylogenetic gap. Obtained sequences grouped closely with CA XIII in phylogenetic analyses. CA II mRNA transcripts were also found in erythrocytes, but found at less than half the levels of CA XIII. Structural analysis suggested similar biochemical activity as the respective mammalian isoforms, with CA XIII being a low activity isoform. Biochemical characterization verified that the majority of CA activity in the erythrocytes was due to a high activity CA II-like isoform; however, titration with copper supported the presence of two CA pools. The CA II-like pool accounted for 90 % of the total activity. To assess potential disparate roles of these isoforms a feeding stress was used to up-regulate CO2 excretion pathways. Significant up-regulation of CA II and the anion exchanger was observed; CA XIII was strongly down-regulated. While these results do not provide insight into the role of CA XIII in the erythrocytes, they do suggest that the presence of two isoforms is not simply a case of physiological redundancy. Copyright © 2015. Published by Elsevier Inc.

Proteomic analysis reveals APC-dependent post-translational modifications and identifies a novel regulator of β-catenin.

PubMed

Blundon, Malachi A; Schlesinger, Danielle R; Parthasarathy, Amritha; Smith, Samantha L; Kolev, Hannah M; Vinson, David A; Kunttas-Tatli, Ezgi; McCartney, Brooke M; Minden, Jonathan S

2016-07-15

Wnt signaling generates patterns in all embryos, from flies to humans, and controls cell fate, proliferation and metabolic homeostasis. Inappropriate Wnt pathway activation results in diseases, including colorectal cancer. The adenomatous polyposis coli (APC) tumor suppressor gene encodes a multifunctional protein that is an essential regulator of Wnt signaling and cytoskeletal organization. Although progress has been made in defining the role of APC in a normal cellular context, there are still significant gaps in our understanding of APC-dependent cellular function and dysfunction. We expanded the APC-associated protein network using a combination of genetics and a proteomic technique called two-dimensional difference gel electrophoresis (2D-DIGE). We show that loss of Drosophila Apc2 causes protein isoform changes reflecting misregulation of post-translational modifications (PTMs), which are not dependent on β-catenin transcriptional activity. Mass spectrometry revealed that proteins involved in metabolic and biosynthetic pathways, protein synthesis and degradation, and cell signaling are affected by Apc2 loss. We demonstrate that changes in phosphorylation partially account for the altered PTMs in APC mutants, suggesting that APC mutants affect other types of PTM. Finally, through this approach Aminopeptidase P was identified as a new regulator of β-catenin abundance in Drosophila embryos. This study provides new perspectives on the cellular effects of APC that might lead to a deeper understanding of its role in development. © 2016. Published by The Company of Biologists Ltd.

Finding Protein and Nucleotide Similarities with FASTA

PubMed Central

Pearson, William R.

2016-01-01

The FASTA programs provide a comprehensive set of rapid similarity searching tools ( fasta36, fastx36, tfastx36, fasty36, tfasty36), similar to those provided by the BLAST package, as well as programs for slower, optimal, local and global similarity searches ( ssearch36, ggsearch36) and for searching with short peptides and oligonucleotides ( fasts36, fastm36). The FASTA programs use an empirical strategy for estimating statistical significance that accommodates a range of similarity scoring matrices and gap penalties, improving alignment boundary accuracy and search sensitivity (Unit 3.5). The FASTA programs can produce “BLAST-like” alignment and tabular output, for ease of integration into existing analysis pipelines, and can search small, representative databases, and then report results for a larger set of sequences, using links from the smaller dataset. The FASTA programs work with a wide variety of database formats, including mySQL and postgreSQL databases (Unit 9.4). The programs also provide a strategy for integrating domain and active site annotations into alignments and highlighting the mutational state of functionally critical residues. These protocols describe how to use the FASTA programs to characterize protein and DNA sequences, using protein:protein, protein:DNA, and DNA:DNA comparisons. PMID:27010337

Cryptococcal titan cell formation is regulated by G-protein signaling in response to multiple stimuli.

PubMed

Okagaki, Laura H; Wang, Yina; Ballou, Elizabeth R; O'Meara, Teresa R; Bahn, Yong-Sun; Alspaugh, J Andrew; Xue, Chaoyang; Nielsen, Kirsten

2011-10-01

The titan cell is a recently described morphological form of the pathogenic fungus Cryptococcus neoformans. Occurring during the earliest stages of lung infection, titan cells are 5 to 10 times larger than the normal yeast-like cells, thereby resisting engulfment by lung phagocytes and favoring the persistence of infection. These enlarged cells exhibit an altered capsule structure, a thickened cell wall, increased ploidy, and resistance to nitrosative and oxidative stresses. We demonstrate that two G-protein-coupled receptors are important for induction of the titan cell phenotype: the Ste3a pheromone receptor (in mating type a cells) and the Gpr5 protein. Both receptors control titan cell formation through elements of the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. This conserved signaling pathway, in turn, mediates its effect on titan cells through the PKA-regulated Rim101 transcription factor. Additional downstream effectors required for titan cell formation include the G(1) cyclin Pcl103, the Rho104 GTPase, and two GTPase-activating proteins, Gap1 and Cnc1560. These observations support developing models in which the PKA signaling pathway coordinately regulates many virulence-associated phenotypes in diverse human pathogens.

Cryptococcal Titan Cell Formation Is Regulated by G-Protein Signaling in Response to Multiple Stimuli▿†

PubMed Central

Okagaki, Laura H.; Wang, Yina; Ballou, Elizabeth R.; O'Meara, Teresa R.; Bahn, Yong-Sun; Alspaugh, J. Andrew; Xue, Chaoyang; Nielsen, Kirsten

2011-01-01

The titan cell is a recently described morphological form of the pathogenic fungus Cryptococcus neoformans. Occurring during the earliest stages of lung infection, titan cells are 5 to 10 times larger than the normal yeast-like cells, thereby resisting engulfment by lung phagocytes and favoring the persistence of infection. These enlarged cells exhibit an altered capsule structure, a thickened cell wall, increased ploidy, and resistance to nitrosative and oxidative stresses. We demonstrate that two G-protein-coupled receptors are important for induction of the titan cell phenotype: the Ste3a pheromone receptor (in mating type a cells) and the Gpr5 protein. Both receptors control titan cell formation through elements of the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. This conserved signaling pathway, in turn, mediates its effect on titan cells through the PKA-regulated Rim101 transcription factor. Additional downstream effectors required for titan cell formation include the G1 cyclin Pcl103, the Rho104 GTPase, and two GTPase-activating proteins, Gap1 and Cnc1560. These observations support developing models in which the PKA signaling pathway coordinately regulates many virulence-associated phenotypes in diverse human pathogens. PMID:21821718

Immune evasion in ebolavirus infections.

PubMed

Audet, Jonathan; Kobinger, Gary P

2015-02-01

Ebola virus (EBOV) infects humans as well as several animal species. It can lead to a highly lethal disease, with mortality rates approaching 90% in primates. Recent advances have deepened our understanding of how this virus is able to prevent the development of protective immune responses. The EBOV genome encodes eight proteins, four of which were shown to interact with the host in ways that counteract the immune response. The viral protein 35 (VP35) is capable of capping dsRNA and interacts with IRF7 to prevent detection of the virus by immune cells. The main role of the soluble glycoprotein (sGP) is still unclear, but it is capable of subverting the anti-GP1,2 antibody response. The GP1,2 protein has shown anti-tetherin activity and the ability to hide cell-surface proteins. Finally, VP24 interferes with the production of interferons (IFNs) and with IFN signaling in infected cells. Taken together, these data point to extensive adaptation of EBOV to evade the immune system of dead end hosts. While our understanding of the interactions between the human and viral proteins increases, details of those interactions in other hosts remain largely unclear and represent a gap in our knowledge.

Finding Protein and Nucleotide Similarities with FASTA.

PubMed

Pearson, William R

2016-03-24

The FASTA programs provide a comprehensive set of rapid similarity searching tools (fasta36, fastx36, tfastx36, fasty36, tfasty36), similar to those provided by the BLAST package, as well as programs for slower, optimal, local, and global similarity searches (ssearch36, ggsearch36), and for searching with short peptides and oligonucleotides (fasts36, fastm36). The FASTA programs use an empirical strategy for estimating statistical significance that accommodates a range of similarity scoring matrices and gap penalties, improving alignment boundary accuracy and search sensitivity. The FASTA programs can produce "BLAST-like" alignment and tabular output, for ease of integration into existing analysis pipelines, and can search small, representative databases, and then report results for a larger set of sequences, using links from the smaller dataset. The FASTA programs work with a wide variety of database formats, including mySQL and postgreSQL databases. The programs also provide a strategy for integrating domain and active site annotations into alignments and highlighting the mutational state of functionally critical residues. These protocols describe how to use the FASTA programs to characterize protein and DNA sequences, using protein:protein, protein:DNA, and DNA:DNA comparisons. Copyright © 2016 John Wiley & Sons, Inc.

Biogeoscience from a Metallomic and Proteomic Perspective

NASA Astrophysics Data System (ADS)

Anbar, A. D.; Shock, E.

2004-12-01

In the wake of the genomics revolution, life scientists are expanding their focus from the genome to the "proteome" - the assemblage of all proteins in a cell - and the "metallome" - the distribution of inorganic species in a cell. The proteome and metallome are tightly connected because proteins and protein products are intimately involved in the transport and homeostasis of inorganic elements, and because many enzymes depend on inorganic elements for catalytic activity. Together, they are at the heart of metabolic function. Unlike the relatively static genome, the proteome and metallome are extremely dynamic, changing rapidly in response to environmental cues. They are substantially more complex than the genome; for example, in humans, some 30,000 genes code for approximately 500,000 proteins. Metaphorically, the proteome and metallome constitute the complex, dynamic "language" by which the genome and the environment communicate. Therefore biogeochemists, like life scientists, are moving beyond a strictly genomic perspective. Research guided by proteomic and metallomic perspectives and methodologies should provide new insights into the connections between life and the inorganic Earth in modern environments, and the evolution of these connections through time. For example, biogeochemical research in modern environments, such as Yellowstone hot springs, is hindered by the gap between genomic determinations of metabolic potential in ecosystems and geochemical characterizations of the energetic boundary conditions faced by these ecosystems; genomics tells us "who is there" and geochemistry tells us "what they might be doing", but neither genomics nor geochemistry easily provide quantitative information about which metabolisms are actually active or a framework for understanding why ecosystems do not fully exploit the energy available in their surroundings. Such questions are fundamentally kinetic rather than thermodynamic and therefore demand that we characterize and understand the proteins and inorganic elements used by organisms to catalyze reactions and capture energy from their surroundings. Similar challenges are faced when attempting to map the evolutionary relationships inferred from phylogenetic analyses of genomes to ecological histories determined by geochemists and paleobiologists - for example, ongoing efforts to understand the evolutionary history of eukaryotes and metazoa - because the driving forces for the evolution and ecological radiation of organisms lie at the intersection of metabolism and environment, and hence in the gap between genomes and geochemistry. Future progress in understanding the biogeochemistry of modern and ancient environments will be spurred by integrating proteomic and metallomic methods and perspectives.

Distal gap junctions and active dendrites can tune network dynamics.

PubMed

Saraga, Fernanda; Ng, Leo; Skinner, Frances K

2006-03-01

Gap junctions allow direct electrical communication between CNS neurons. From theoretical and modeling studies, it is well known that although gap junctions can act to synchronize network output, they can also give rise to many other dynamic patterns including antiphase and other phase-locked states. The particular network pattern that arises depends on cellular, intrinsic properties that affect firing frequencies as well as the strength and location of the gap junctions. Interneurons or GABAergic neurons in hippocampus are diverse in their cellular characteristics and have been shown to have active dendrites. Furthermore, parvalbumin-positive GABAergic neurons, also known as basket cells, can contact one another via gap junctions on their distal dendrites. Using two-cell network models, we explore how distal electrical connections affect network output. We build multi-compartment models of hippocampal basket cells using NEURON and endow them with varying amounts of active dendrites. Two-cell networks of these model cells as well as reduced versions are explored. The relationship between intrinsic frequency and the level of active dendrites allows us to define three regions based on what sort of network dynamics occur with distal gap junction coupling. Weak coupling theory is used to predict the delineation of these regions as well as examination of phase response curves and distal dendritic polarization levels. We find that a nonmonotonic dependence of network dynamic characteristics (phase lags) on gap junction conductance occurs. This suggests that distal electrical coupling and active dendrite levels can control how sensitive network dynamics are to gap junction modulation. With the extended geometry, gap junctions located at more distal locations must have larger conductances for pure synchrony to occur. Furthermore, based on simulations with heterogeneous networks, it may be that one requires active dendrites if phase-locking is to occur in networks formed with distal gap junctions.

Attentional Capacity Limits Gap Detection during Concurrent Sound Segregation.

PubMed

Leung, Ada W S; Jolicoeur, Pierre; Alain, Claude

2015-11-01

Detecting a brief silent interval (i.e., a gap) is more difficult when listeners perceive two concurrent sounds rather than one in a sound containing a mistuned harmonic in otherwise in-tune harmonics. This impairment in gap detection may reflect the interaction of low-level encoding or the division of attention between two sound objects, both of which could interfere with signal detection. To distinguish between these two alternatives, we compared ERPs during active and passive listening with complex harmonic tones that could include a gap, a mistuned harmonic, both features, or neither. During active listening, participants indicated whether they heard a gap irrespective of mistuning. During passive listening, participants watched a subtitled muted movie of their choice while the same sounds were presented. Gap detection was impaired when the complex sounds included a mistuned harmonic that popped out as a separate object. The ERP analysis revealed an early gap-related activity that was little affected by mistuning during the active or passive listening condition. However, during active listening, there was a marked decrease in the late positive wave that was thought to index attention and response-related processes. These results suggest that the limitation in detecting the gap is related to attentional processing, possibly divided attention induced by the concurrent sound objects, rather than deficits in preattentional sensory encoding.

Developmental and Adult GAP-43 Deficiency in Mice Dynamically Alters Hippocampal Neurogenesis and Mossy Fiber Volume

PubMed Central

Latchney, Sarah E.; Masiulis, Irene; Zaccaria, Kimberly J.; Lagace, Diane C.; Powell, Craig M.; McCasland, James S.; Eisch, Amelia J.

2014-01-01

Growth Associated Protein-43 (GAP-43) is a pre-synaptic protein that plays key roles in axonal growth and guidance and in modulating synapse formation. Previous work has demonstrated that mice lacking one allele of this gene [GAP-43(+/-) mice] exhibit hippocampal structural abnormalities and impaired spatial learning and stress-induced behavioral withdrawal and anxiety (Zaccaria et al., 2010), behaviors that are dependent on proper hippocampal circuitry and function. Given the correlation between hippocampal function, synaptic connectivity, and neurogenesis, we tested if behaviorally-naïve GAP-43(+/-) mice had alterations in either neurogenesis or synaptic connectivity in the hippocampus during early postnatal development and young adulthood, and following behavior testing in older adults. To test our hypothesis, we examined hippocampal cell proliferation (Ki67), number of immature neuroblasts (DCX), and mossy fiber volume (synaptoporin) in behaviorally-naïve postnatal (P) day 9 (P9), P26, and behaviorally-experienced 5-7 month old GAP-43(+/-) and (+/+) littermate mice. P9 GAP-43(+/-) mice had fewer Ki67+ and DCX+ cells compared to (+/+) mice, particularly in the posterior dentate gyrus, and smaller mossy fiber volume in the same region. In young adulthood, however, male GAP-43(+/-) mice had more Ki67+ and DCX+ cells and greater mossy fiber volume in the posterior dentate gyrus relative to male (+/+). These increases were not seen in females. In 5-7 month old GAP-43(+/-) mice whose behaviors were the focus of our prior publication (Zaccaria et al., 2010), there was no global change in number of proliferating or immature neurons relative to (+/+) mice. However, more detailed analysis revealed fewer proliferative DCX+ cells in the anterior dentate gyrus of male GAP-43(+/-) mice compared to male (+/+) mice. This reduction was not observed in females. These results suggest that young GAP-43(+/-) mice have decreased hippocampal neurogenesis and synaptic connectivity, but slightly older mice have greater hippocampal neurogenesis and synaptic connectivity. In conjunction with our previous study, these findings suggest GAP-43 is dynamically involved in early postnatal and adult hippocampal neurogenesis and synaptic connectivity, possibly contributing to the GAP-43(+/-) behavioral phenotype. PMID:24576816

Disordered nucleiome: Abundance of intrinsic disorder in the DNA- and RNA-binding proteins in 1121 species from Eukaryota, Bacteria and Archaea.

PubMed

Wang, Chen; Uversky, Vladimir N; Kurgan, Lukasz

2016-05-01

Intrinsically disordered proteins (IDPs) are abundant in various proteomes, where they play numerous important roles and complement biological activities of ordered proteins. Among functions assigned to IDPs are interactions with nucleic acids. However, often, such assignments are made based on the guilty-by-association principle. The validity of the extension of these correlations to all nucleic acid binding proteins has never been analyzed on a large scale across all domains of life. To fill this gap, we perform a comprehensive computational analysis of the abundance of intrinsic disorder and intrinsically disordered domains in nucleiomes (∼548 000 nucleic acid binding proteins) of 1121 species from Archaea, Bacteria and Eukaryota. Nucleiome is a whole complement of proteins involved in interactions with nucleic acids. We show that relative to other proteins in the corresponding proteomes, the DNA-binding proteins have significantly increased disorder content and are significantly enriched in disordered domains in Eukaryotes but not in Archaea and Bacteria. The RNA-binding proteins are significantly enriched in the disordered domains in Bacteria, Archaea and Eukaryota, while the overall abundance of disorder in these proteins is significantly increased in Bacteria, Archaea, animals and fungi. The high abundance of disorder in nucleiomes supports the notion that the nucleic acid binding proteins often require intrinsic disorder for their functions and regulation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

G protein-coupled receptors: bridging the gap from the extracellular signals to the Hippo pathway.

PubMed

Zhou, Xin; Wang, Zhen; Huang, Wei; Lei, Qun-Ying

2015-01-01

The Hippo pathway is crucial in organ size control, whereas its dysregulation contributes to organ degeneration or tumorigenesis. The kinase cascade of MST1/2 and LATS1/2 and the coupling transcription co-activators YAP/TAZ represent the core components of the Hippo pathway. Extensive studies have identified a number of upstream regulators of the Hippo pathway, including contact inhibition, mechanic stress, extracellular matrix stiffness, cytoskeletal rearrangement, and some molecules of cell polarity and cell junction. However, how the diffuse extracellular signals regulate the Hippo pathway puzzles the researchers for a long time. Unexpectedly, recent elegant studies demonstrated that stimulation of some G protein-coupled receptors (GPCRs), such as lysophosphatidic acid receptor, sphingosine-1-phosphate receptor, and the protease activated receptor PAR1, causes potent YAP/TAZ dephosphorylation and activation by promoting actin cytoskeleton assemble. In this review, we briefly describe the components of the Hippo pathway and focus on the recent progress with respect to the regulation of the Hippo pathway by GPCRs and G proteins in cancer cells. In addition, we also discuss the potential therapeutic roles targeting the Hippo pathway in human cancers. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Single-molecule analysis of diffusion and trapping of STIM1 and Orai1 at endoplasmic reticulum–plasma membrane junctions

PubMed Central

Wu, Minnie M.; Covington, Elizabeth D.; Lewis, Richard S.

2014-01-01

Following endoplasmic reticulum (ER) Ca2+ depletion, STIM1 and Orai1 complexes assemble autonomously at ER–plasma membrane (PM) junctions to trigger store-operated Ca2+ influx. One hypothesis to explain this process is a diffusion trap in which activated STIM1 diffusing in the ER becomes trapped at junctions through interactions with the PM, and STIM1 then traps Orai1 in the PM through binding of its calcium release-activated calcium activation domain. We tested this model by analyzing STIM1 and Orai1 diffusion using single-particle tracking, photoactivation of protein ensembles, and Monte Carlo simulations. In resting cells, STIM1 diffusion is Brownian, while Orai1 is slightly subdiffusive. After store depletion, both proteins slow to the same speeds, consistent with complex formation, and are confined to a corral similar in size to ER–PM junctions. While the escape probability at high STIM:Orai expression ratios is <1%, it is significantly increased by reducing the affinity of STIM1 for Orai1 or by expressing the two proteins at comparable levels. Our results provide direct evidence that STIM-Orai complexes are trapped by their physical connections across the junctional gap, but also reveal that the complexes are surprisingly dynamic, suggesting that readily reversible binding reactions generate free STIM1 and Orai1, which engage in constant diffusional exchange with extrajunctional pools. PMID:25057023

Carbon Tetrachloride at Hepatotoxic Levels Blocks Reversibly Gap Junctions between Rat Hepatocytes

NASA Astrophysics Data System (ADS)

Saez, J. C.; Bennett, M. V. L.; Spray, D. C.

1987-05-01

Electrical coupling and dye coupling between pairs of rat hepatocytes were reversibly reduced by brief exposure to halogenated methanes (CBrCl3, CCl4, and CHCl3). The potency of different halomethanes in uncoupling hepatocytes was comparable to their hepatotoxicity in vivo, and the rank order was the same as that of their tendency to form free radicals. The effect of carbon tetrachloride (CCl4) on hepatocytes was substantially reduced by prior treatment with SKF 525A, an inhibitor of cytochrome P-450, and by exposure to the reducing reagent β -mercaptoethanol. Halomethane uncoupling occurred with or without extracellular calcium and did not change intracellular concentrations of calcium and hydrogen ions or the phosphorylation state of the main gap-junctional protein. Thus the uncoupling appears to depend on cytochrome P-450 oxidative metabolism in which free radicals are generated and may result from oxidation of the gap-junctional protein or of a regulatory molecule that leads to closure of gap-junctional channels. Decreases in junctional conductance may be a rapid cellular response to injury that protects healthy cells by uncoupling them from unhealthy ones.

Mitogen-Activated Protein Kinase Phosphatase-2 Deletion Impairs Synaptic Plasticity and Hippocampal-Dependent Memory.

PubMed

Abdul Rahman, Nor Zaihana; Greenwood, Sam M; Brett, Ros R; Tossell, Kyoko; Ungless, Mark A; Plevin, Robin; Bushell, Trevor J

2016-02-24

Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer's disease. Thus, there is great interest in understanding the signaling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system, and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2(-/-) mice), we show that long-term potentiation is impaired in MKP-2(-/-) mice compared with MKP-2(+/+) controls whereas neuronal excitability, evoked synaptic transmission, and paired-pulse facilitation remain unaltered. Furthermore, spontaneous EPSC (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2(-/-) mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures, which may account for the increase in sEPSC frequency. In addition, no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2(-/-) mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signaling. Copyright © 2016 Abdul Rahman et al.

Altered glutamyl-aminopeptidase activity and expression in renal neoplasms

PubMed Central

2014-01-01

Background Advances in the knowledge of renal neoplasms have demonstrated the implication of several proteases in their genesis, growth and dissemination. Glutamyl-aminopeptidase (GAP) (EC. 3.4.11.7) is a zinc metallopeptidase with angiotensinase activity highly expressed in kidney tissues and its expression and activity have been associated wtih tumour development. Methods In this prospective study, GAP spectrofluorometric activity and immunohistochemical expression were analysed in clear-cell (CCRCC), papillary (PRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytoma (RO). Data obtained in tumour tissue were compared with those from the surrounding uninvolved kidney tissue. In CCRCC, classic pathological parameters such as grade, stage and tumour size were stratified following GAP data and analyzed for 5-year survival. Results GAP activity in both the membrane-bound and soluble fractions was sharply decreased and its immunohistochemical expression showed mild staining in the four histological types of renal tumours. Soluble and membrane-bound GAP activities correlated with tumour grade and size in CCRCCs. Conclusions This study suggests a role for GAP in the neoplastic development of renal tumours and provides additional data for considering the activity and expression of this enzyme of interest in the diagnosis and prognosis of renal neoplasms. PMID:24885240

Alpinia oxyphylla Miq. fruit extract activates IGFR-PI3K/Akt signaling to induce Schwann cell proliferation and sciatic nerve regeneration.

PubMed

Chang, Yung-Ming; Chang, Hen-Hong; Tsai, Chin-Chuan; Lin, Hung-Jen; Ho, Tsung-Jung; Ye, Chi-Xin; Chiu, Ping-Ling; Chen, Yueh-Sheng; Chen, Ray-Jade; Huang, Chih-Yang; Lin, Chien-Chung

2017-03-31

It is known that the medicinal herb Alpinia oxyphylla Miq. is widely used as a remedy for diarrhea as well as the symptoms accompanying hypertension and cerebrovascular disorders. Moreover, it has also been reported that Alpinia oxyphylla Miq. has beneficial effects on anti-senescence and neuro-protection. This study focuses on the molecular mechanisms by which the Alpinia oxyphylla Miq. fruits promote neuron regeneration. A piece of silicone rubber was guided across a 15 mm gap in the sciatic nerve of a rat. This nerve gap was then filled with various doses of Alpinia oxyphylla Miq. fruits to assess their regenerative effect on damaged nerves. Further, we investigated the role of Alpinia oxyphylla Miq. fruits in RSC96 Schwann cell proliferation. Our current results showed that treatment with the extract of Alpinia oxyphylla Miq. fruits triggers the phosphorylated insulin-like growth factor-1 receptor- phosphatidylinositol 3-kinase/serine-threonine kinase pathway, and up-regulated the proliferating cell nuclear antigen in a dose-dependent manner. Cell cycle analysis on RSC96 Schwann cells showed that, after exposure to Alpinia oxyphylla Miq. fruit extract, the transition from the first gap phase to the synthesis phase occurs in 12-18 h. The expression of the cell cycle regulatory proteins cyclin D1, cyclin E and cyclin A increased in a dose-dependent manner. Transfection with a small interfering RNA blocked the expression of phosphatidylinositol 3-kinase and induced down-regulation both on the mRNA and protein levels, which resulted in a reduction of the expression of the survival factor B-cell lymphoma 2. We provide positive results that demonstrate that Alpinia oxyphylla Miq. fruits facilitate the survival and proliferation of RSC96 cells via insulin-like growth factor-1 signaling.

Astragaloside IV Attenuates Glutamate-Induced Neurotoxicity in PC12 Cells through Raf-MEK-ERK Pathway.

PubMed

Yue, Rongcai; Li, Xia; Chen, Bingyang; Zhao, Jing; He, Weiwei; Yuan, Hu; Yuan, Xing; Gao, Na; Wu, Guozhen; Jin, Huizi; Shan, Lei; Zhang, Weidong

2015-01-01

Astragaloside IV (AGS-IV) is a main active ingredient of Astragalus membranaceus Bunge, a medicinal herb prescribed as an immunostimulant, hepatoprotective, antiperspirant, a diuretic or a tonic as documented in Chinese Materia Medica. In the present study, we employed a high-throughput comparative proteomic approach based on 2D-nano-LC-MS/MS to investigate the possible mechanism of action involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. Differential proteins were identified, among which 13 proteins survived the stringent filter criteria and were further included for functional discussion. Two proteins (vimentin and Gap43) were randomly selected, and their expression levels were further confirmed by western blots analysis. The results matched well with those of proteomics. Furthermore, network analysis of protein-protein interactions (PPI) and pathways enrichment with AGS-IV associated proteins were carried out to illustrate its underlying molecular mechanism. Proteins associated with signal transduction, immune system, signaling molecules and interaction, and energy metabolism play important roles in neuroprotective effect of AGS-IV and Raf-MEK-ERK pathway was involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. This study demonstrates that comparative proteomics based on shotgun approach is a valuable tool for molecular mechanism studies, since it allows the simultaneously evaluate the global proteins alterations.

MTV1 and MTV4 encode plant-specific ENTH and ARF GAP proteins that mediate clathrin-dependent trafficking of vacuolar cargo from the trans-Golgi network.

PubMed

Sauer, Michael; Delgadillo, M Otilia; Zouhar, Jan; Reynolds, Gregory D; Pennington, Janice G; Jiang, Liwen; Liljegren, Sarah J; Stierhof, York-Dieter; De Jaeger, Geert; Otegui, Marisa S; Bednarek, Sebastian Y; Rojo, Enrique

2013-06-01

Many soluble proteins transit through the trans-Golgi network (TGN) and the prevacuolar compartment (PVC) en route to the vacuole, but our mechanistic understanding of this vectorial trafficking step in plants is limited. In particular, it is unknown whether clathrin-coated vesicles (CCVs) participate in this transport step. Through a screen for modified transport to the vacuole (mtv) mutants that secrete the vacuolar protein VAC2, we identified MTV1, which encodes an epsin N-terminal homology protein, and MTV4, which encodes the ADP ribosylation factor GTPase-activating protein nevershed/AGD5. MTV1 and NEV/AGD5 have overlapping expression patterns and interact genetically to transport vacuolar cargo and promote plant growth, but they have no apparent roles in protein secretion or endocytosis. MTV1 and NEV/AGD5 colocalize with clathrin at the TGN and are incorporated into CCVs. Importantly, mtv1 nev/agd5 double mutants show altered subcellular distribution of CCV cargo exported from the TGN. Moreover, MTV1 binds clathrin in vitro, and NEV/AGD5 associates in vivo with clathrin, directly linking these proteins to CCV formation. These results indicate that MTV1 and NEV/AGD5 are key effectors for CCV-mediated trafficking of vacuolar proteins from the TGN to the PVC in plants.

Innexin2 gap junctions in somatic support cells are required for cyst formation and for egg chamber formation in Drosophila.

PubMed

Mukai, Masanori; Kato, Hirotaka; Hira, Seiji; Nakamura, Katsuhiro; Kita, Hiroaki; Kobayashi, Satoru

2011-01-01

Germ cells require intimate associations with surrounding somatic cells during gametogenesis. During oogenesis, gap junctions mediate communication between germ cells and somatic support cells. However, the molecular mechanisms by which gap junctions regulate the developmental processes during oogenesis are poorly understood. We have identified a female sterile allele of innexin2 (inx2), which encodes a gap junction protein in Drosophila. In females bearing this inx2 allele, cyst formation and egg chamber formation are impaired. In wild-type germaria, Inx2 is strongly expressed in escort cells and follicle cells, both of which make close contact with germline cells. We show that inx2 function in germarial somatic cells is required for the survival of early germ cells and promotes cyst formation, probably downstream of EGFR pathway, and that inx2 function in follicle cells promotes egg chamber formation through the regulation of DE-cadherin and Bazooka (Baz) at the boundary between germ cells and follicle cells. Furthermore, genetic experiments demonstrate that inx2 interacts with the zero population growth (zpg) gene, which encodes a germline-specific gap junction protein. These results indicate a multifunctional role for Inx2 gap junctions in somatic support cells in the regulation of early germ cell survival, cyst formation and egg chamber formation. Inx2 gap junctions may mediate the transfer of nutrients and signal molecules between germ cells and somatic support cells, as well as play a role in the regulation of cell adhesion. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Structural basis for the binding of tryptophan-based motifs by δ-COP

PubMed Central

Suckling, Richard J.; Poon, Pak Phi; Travis, Sophie M.; Majoul, Irina V.; Hughson, Frederick M.; Evans, Philip R.; Duden, Rainer; Owen, David J.

2015-01-01

Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ’ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1–6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing. PMID:26578768

BAC-recombineering for studying plant gene regulation: developmental control and cellular localization of SnRK1 kinase subunits.

PubMed

Bitrián, Marta; Roodbarkelari, Farshad; Horváth, Mihály; Koncz, Csaba

2011-03-01

Recombineering, permitting precise modification of genes within bacterial artificial chromosomes (BACs) through homologous recombination mediated by lambda phage-encoded Red proteins, is a widely used powerful tool in mouse, Caenorhabditis and Drosophila genetics. As Agrobacterium-mediated transfer of large DNA inserts from binary BACs and TACs into plants occurs at low frequency, recombineering is so far seldom exploited in the analysis of plant gene functions. We have constructed binary plant transformation vectors, which are suitable for gap-repair cloning of genes from BACs using recombineering methods previously developed for other organisms. Here we show that recombineering facilitates PCR-based generation of precise translational fusions between coding sequences of fluorescent reporter and plant proteins using galK-based exchange recombination. The modified target genes alone or as part of a larger gene cluster can be transferred by high-frequency gap-repair into plant transformation vectors, stably maintained in Agrobacterium and transformed without alteration into plants. Versatile application of plant BAC-recombineering is illustrated by the analysis of developmental regulation and cellular localization of interacting AKIN10 catalytic and SNF4 activating subunits of Arabidopsis Snf1-related (SnRK1) protein kinase using in vivo imaging. To validate full functionality and in vivo interaction of tagged SnRK1 subunits, it is demonstrated that immunoprecipitated SNF4-YFP is bound to a kinase that phosphorylates SnRK1 candidate substrates, and that the GFP- and YFP-tagged kinase subunits co-immunoprecipitate with endogenous wild type AKIN10 and SNF4. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

30 CFR 285.811 - When must I follow my Safety Management System?

Code of Federal Regulations, 2011 CFR

2011-07-01

... Activities Conducted Under SAPs, COPs and GAPs Safety Management Systems § 285.811 When must I follow my... activities described in your approved COP, SAP, or GAP. You must conduct all activities described in your approved COP, SAP, or GAP in accordance with the Safety Management System you described, as required by...

The prescriptions from Shenghui soup enhanced neurite growth and GAP-43 expression level in PC12 cells.

PubMed

Zhang, Qi; Zhang, Zi-Jian; Wang, Xing-Hua; Ma, Jie; Song, Yue-Han; Liang, Mi; Lin, Sen-Xiang; Zhao, Jie; Zhang, Ao-Zhe; Li, Feng; Hua, Qian

2016-09-20

Shenghui soup is a traditional Chinese herbal medicine used in clinic for the treatment of forgetfulness. In order to understanding the prescription principle, the effects of "tonifying qi and strengthening spleen" group (TQSS) including Poria cocos (Schw.) Wolf. and Panax ginseng C.A.Mey and "eliminating phlegm and strengthening intelligence" group (EPSI) composed of Polygala tenuifolia Willd., Acorus calamus L. and Sinapis alba L from the herb complex on neurite growth in PC12 cells, two disassembled prescriptions derived from Shenghui soup and their molecular mechanisms were investigated. Firstly, CCK-8 kit was used to detect the impact of the two prescriptions on PC12 cell viability; and Flow cytometry was performed to measure the cell apoptosis when PC12 cells were treated with these drugs. Secondly, the effect of the two prescriptions on the differentiation of PC12 cells was observed. Finally, the mRNA and protein expression levels of GAP-43 were analyzed by RT-PCR and western blot, respectively. "Tonifying qi and strengthening spleen" prescription decreased cell viability in a dose-dependent manner, but had no significant effect on cell apoptosis. Meanwhile, it could improve neurite growth and elevate the mRNA and protein expression level of GAP-43. "Eliminating phlegm and strengthening intelligence" prescription also exerted the similar effects on cell viability and apoptosis. Furthermore, it could also enhance cell neurite growth, with a higher expression level of GAP-43 mRNA and protein. "Tonifying qi and strengthening spleen" and "eliminating phlegm and strengthening intelligence" prescriptions from Shenghui soup have a positive effect on neurite growth. Their effects are related to the up-regulating expression of GAP-43.

Proteomic analysis of the crayfish gastrolith chitinous extracellular matrix reveals putative protein complexes and a central role for GAP 65.

PubMed

Glazer, Lilah; Roth, Ziv; Weil, Simy; Aflalo, Eliahu D; Khalaila, Isam; Sagi, Amir

2015-10-14

Chitin is a major component of arthropod cuticles, where it forms a three-dimensional network that constitutes the scaffold upon which cuticles form. The chitin fibers that form this network are closely associated with specific structural proteins, while the cuticular matrix contains many additional structural, enzymatic and other proteins. We study the crayfish gastrolith as a simple model for the assembly of calcified cuticular structures, with particular focus on the proteins involved in this process. The present study integrates a gastrolith-forming epithelium transcriptomic library with data from mass spectrometry analysis of proteins extracted from the gastrolith matrix to obtain a near-complete picture of gastrolith protein content. Using native protein separation we identified 24 matrix proteins, of which 14 are novel. Further analysis led to discovery of three putative protein complexes, all containing GAP 65 the most abundant gastrolith structural protein. Using immunological methods we further studied the role of GAP 65 in the gastrolith matrix and forming epithelium, as well as in the newly identified protein complexes. We propose that gastrolith matrix construction is a sequential process in which protein complexes are dynamically assembled and disassembled around GAP 65, thus changing their functional properties to perform each step in the construction process. The scientific interest on which this study is based arises from three main features of gastroliths: (1) Gastroliths possess partial analogy to cuticles both in structural and molecular properties, and may be regarded, with the appropriate reservations (see Introduction), as simple models for cuticle assembly. At the same time, gastroliths are terminally assembled during a well-defined period, which can be controlled in the laboratory, making them significantly easier to study than cuticles. (2) Gastroliths, like the crayfish exoskeleton, contain stable amorphous calcium carbonate (ACC) rather than crystalline calcite. The biological mechanism for the stabilization of a naturally unstable, but at the same time biologically highly available, calcium carbonate polymorph is of great interest from the pharmaceutical point of view. (3) The gastrolith organic matrix is based on a highly structured chitin network that interacts with a variety of substances. This biologically manipulated, biodegradable structure is in itself of biotechnological and pharmaceutical potential. A growing body of evidence indicates that proteins play central roles in all above aspects of gastrolith construction. This study offers the first comprehensive screening of gastrolith proteins, and we believe that the analysis presented in this work can not only help reveal basic biological questions regarding assembly of mineralized and non-mineralized cuticular structures, but may also serve as basis for applied research in the fields of agriculture (e.g. cuticle-based pest management), health (e.g. bioavailable calcium supplements and biodegradable drug carriers) and materials science (e.g. non-toxic scaffolds for water purification). Copyright © 2015. Published by Elsevier B.V.

Functional Anthology of Intrinsic Disorder. II. Cellular Components, Domains, Technical Terms, Developmental Processes and Coding Sequence Diversities Correlated with Long Disordered Regions

PubMed Central

Vucetic, Slobodan; Xie, Hongbo; Iakoucheva, Lilia M.; Oldfield, Christopher J.; Dunker, A. Keith; Obradovic, Zoran; Uversky, Vladimir N.

2008-01-01

Biologically active proteins without stable ordered structure (i.e., intrinsically disordered proteins) are attracting increased attention. Functional repertoires of ordered and disordered proteins are very different, and the ability to differentiate whether a given function is associated with intrinsic disorder or with a well-folded protein is crucial for modern protein science. However, there is a large gap between the number of proteins experimentally confirmed to be disordered and their actual number in nature. As a result, studies of functional properties of confirmed disordered proteins, while helpful in revealing the functional diversity of protein disorder, provide only a limited view. To overcome this problem, a bioinformatics approach for comprehensive study of functional roles of protein disorder was proposed in the first paper of this series (Xie H., Vucetic S., Iakoucheva L.M., Oldfield C.J., Dunker A.K., Obradovic Z., Uversky V.N. (2006) Functional anthology of intrinsic disorder. I. Biological processes and functions of proteins with long disordered regions. J. Proteome Res.). Applying this novel approach to Swiss-Prot sequences and functional keywords, we found over 238 and 302 keywords to be strongly positively or negatively correlated, respectively, with long intrinsically disordered regions. This paper describes ~90 Swiss-Prot keywords attributed to the cellular components, domains, technical terms, developmental processes and coding sequence diversities possessing strong positive and negative correlation with long disordered regions. PMID:17391015

Functional anthology of intrinsic disorder. 2. Cellular components, domains, technical terms, developmental processes, and coding sequence diversities correlated with long disordered regions.

PubMed

Vucetic, Slobodan; Xie, Hongbo; Iakoucheva, Lilia M; Oldfield, Christopher J; Dunker, A Keith; Obradovic, Zoran; Uversky, Vladimir N

2007-05-01

Biologically active proteins without stable ordered structure (i.e., intrinsically disordered proteins) are attracting increased attention. Functional repertoires of ordered and disordered proteins are very different, and the ability to differentiate whether a given function is associated with intrinsic disorder or with a well-folded protein is crucial for modern protein science. However, there is a large gap between the number of proteins experimentally confirmed to be disordered and their actual number in nature. As a result, studies of functional properties of confirmed disordered proteins, while helpful in revealing the functional diversity of protein disorder, provide only a limited view. To overcome this problem, a bioinformatics approach for comprehensive study of functional roles of protein disorder was proposed in the first paper of this series (Xie, H.; Vucetic, S.; Iakoucheva, L. M.; Oldfield, C. J.; Dunker, A. K.; Obradovic, Z.; Uversky, V. N. Functional anthology of intrinsic disorder. 1. Biological processes and functions of proteins with long disordered regions. J. Proteome Res. 2007, 5, 1882-1898). Applying this novel approach to Swiss-Prot sequences and functional keywords, we found over 238 and 302 keywords to be strongly positively or negatively correlated, respectively, with long intrinsically disordered regions. This paper describes approximately 90 Swiss-Prot keywords attributed to the cellular components, domains, technical terms, developmental processes, and coding sequence diversities possessing strong positive and negative correlation with long disordered regions.

Folding thermodynamics of model four-strand antiparallel beta-sheet proteins.

PubMed Central

Jang, Hyunbum; Hall, Carol K; Zhou, Yaoqi

2002-01-01

The thermodynamic properties for three different types of off-lattice four-strand antiparallel beta-strand protein models interacting via a hybrid Go-type potential have been investigated. Discontinuous molecular dynamic simulations have been performed for different sizes of the bias gap g, an artificial measure of a model protein's preference for its native state. The thermodynamic transition temperatures are obtained by calculating the squared radius of gyration R(g)(2), the root-mean-squared pair separation fluctuation Delta(B), the specific heat C(v), the internal energy of the system E, and the Lindemann disorder parameter Delta(L). Despite these models' simplicity, they exhibit a complex set of protein transitions, consistent with those observed in experimental studies on real proteins. Starting from high temperature, these transitions include a collapse transition, a disordered-to-ordered globule transition, a folding transition, and a liquid-to-solid transition. The high temperature transitions, i.e., the collapse transition and the disordered-to-ordered globule transition, exist for all three beta-strand proteins, although the native-state geometry of the three model proteins is different. However the low temperature transitions, i.e., the folding transition and the liquid-to-solid transition, strongly depend on the native-state geometry of the model proteins and the size of the bias gap. PMID:11806908

Studies of Neurofibromatosis-1 Modifier Genes

DTIC Science & Technology

2005-06-01

inhibitors of GTPase activation by preventing the dissociation of GDP from the inactive GTPase. [E3 DDff The current dogma, at least in the context of...dissociation the cycle is less clear. inhibitors (GD/s), the activity of each of which is potentially modulated in response to various signals. Inactive...function No. of No. of No. of No. of No. of ArfGAPs RabGAPs RapGAPs RasGAPs RhoGAPs BAR IPR004148 Membrane curvature sensor 6 (4) 0 0 0 6 (6) BTK

Release of Native-like Gaseous Proteins from Electrospray Droplets via the Charged Residue Mechanism: Insights from Molecular Dynamics Simulations.

PubMed

McAllister, Robert G; Metwally, Haidy; Sun, Yu; Konermann, Lars

2015-10-07

The mechanism whereby gaseous protein ions are released from charged solvent droplets during electrospray ionization (ESI) remains a matter of debate. Also, it is unclear to what extent electrosprayed proteins retain their solution structure. Molecular dynamics (MD) simulations offer insights into the temporal evolution of protein systems. Surprisingly, there have been no all-atom simulations of the protein ESI process to date. The current work closes this gap by investigating the behavior of protein-containing aqueous nanodroplets that carry excess positive charge. We focus on "native ESI", where proteins initially adopt their biologically active solution structures. ESI proceeds while the protein remains entrapped within the droplet. Protein release into the gas phase occurs upon solvent evaporation to dryness. Droplet shrinkage is accompanied by ejection of charge carriers (Na(+) for the conditions chosen here), keeping the droplet at ∼85% of the Rayleigh limit throughout its life cycle. Any remaining charge carriers bind to the protein as the final solvent molecules evaporate. The outcome of these events is largely independent of the initial protein charge and the mode of charge carrier binding. ESI charge states and collision cross sections of the MD structures agree with experimental data. Our results confirm the Rayleigh/charged residue model (CRM). Field emission of excess Na(+) plays an ancillary role by governing the net charge of the shrinking droplet. Models that envision protein ejection from the droplet are not supported. Most nascent CRM ions retain native-like conformations. For unfolded proteins ESI likely proceeds along routes that are different from the native state mechanism explored here.

Molecular mechanisms regulating formation, trafficking and processing of annular gap junctions.

PubMed

Falk, Matthias M; Bell, Cheryl L; Kells Andrews, Rachael M; Murray, Sandra A

2016-05-24

Internalization of gap junction plaques results in the formation of annular gap junction vesicles. The factors that regulate the coordinated internalization of the gap junction plaques to form annular gap junction vesicles, and the subsequent events involved in annular gap junction processing have only relatively recently been investigated in detail. However it is becoming clear that while annular gap junction vesicles have been demonstrated to be degraded by autophagosomal and endo-lysosomal pathways, they undergo a number of additional processing events. Here, we characterize the morphology of the annular gap junction vesicle and review the current knowledge of the processes involved in their formation, fission, fusion, and degradation. In addition, we address the possibility for connexin protein recycling back to the plasma membrane to contribute to gap junction formation and intercellular communication. Information on gap junction plaque removal from the plasma membrane and the subsequent processing of annular gap junction vesicles is critical to our understanding of cell-cell communication as it relates to events regulating development, cell homeostasis, unstable proliferation of cancer cells, wound healing, changes in the ischemic heart, and many other physiological and pathological cellular phenomena.

The extracellular matrix component laminin promotes gap junction formation in the rat anterior pituitary gland.

PubMed

Horiguchi, Kotaro; Kouki, Tom; Fujiwara, Ken; Kikuchi, Motoshi; Yashiro, Takashi

2011-03-01

Folliculo-stellate (FS) cells in the anterior pituitary gland are believed to have multifunctional properties. FS cells connect to each other not only by mechanical means, but also by gap junctional cell-to-cell communication. Using transgenic rats that express green fluorescent protein (GFP) specifically in FS cells in the anterior pituitary gland (S100b-GFP rats), we recently revealed that FS cells in primary culture markedly change their shape, and form numerous interconnections with neighboring FS cells in the presence of laminin, an extracellular matrix (ECM) component of the basement membrane. Morphological and functional changes in cells are believed to be partly modified by matricrine signaling, by which ECM components function as cellular signals. In the present study, we examined whether gap junction formation between FS cells is affected by matricrine cues. A cell sorter was used to isolate FS cells from male S100b-GFP rat anterior pituitary for primary culture. We observed that mRNA and protein levels of connexin 43 in gap junction channels were clearly higher in the presence of laminin. In addition, we confirmed the formation of gap junctions between FS cells in primary culture by electron microscopy. Interestingly, we also observed that FS cells in the presence of laminin displayed well-developed rough endoplasmic reticulum and Golgi apparatus. Our findings suggest that, in anterior pituitary gland, FS cells may facilitate functional roles such as gap junctional cell-to-cell communication by matricrine signaling.

Active epilepsy prevalence, the treatment gap, and treatment gap risk profile in eastern China: A population-based study.

PubMed

Ding, Xiaoyan; Zheng, Yang; Guo, Yi; Shen, Chunhong; Wang, Shan; Chen, Feng; Yan, Shengqiang; Ding, Meiping

2018-01-01

We measured the prevalence of active epilepsy and investigated the treatment gap and treatment gap risk profile in eastern China. This was a cross-sectional population-based survey conducted in Zhejiang, China, from October 2013 to March 2014. A total 54,976 people were selected using multi-stage cluster sampling. A two-stage questionnaire-based process was used to identify patients with active epilepsy and to record their demographic, socioeconomic, and epilepsy-related features. Logistic regression analysis was used to analyze risk factors of the treatment gap in eastern China, as adjusted for age and sex. We interviewed 50,035 people; 118 had active epilepsy (2.4‰), among which the treatment gap was 58.5%. In multivariate analysis, failure to receive appropriate antiepileptic treatment was associated with higher seizure frequency of 12-23 times per year (adjusted odds ratio=6.874; 95% confidence interval [CI]=2.372-19.918), >24 times per year (adjusted odds ratio=19.623; 95% CI=4.999-77.024), and a lack of health insurance (adjusted odds ratio=7.284; 95% CI=1.321-40.154). Eastern China has relatively lower prevalence of active epilepsy and smaller treatment gap. Interventions aimed at reducing seizure frequency, improving the health insurance system should be investigated as potential targets to further bridge the treatment gap. Copyright © 2017 Elsevier Inc. All rights reserved.

Objective identification of residue ranges for the superposition of protein structures

PubMed Central

2011-01-01

Background The automation of objectively selecting amino acid residue ranges for structure superpositions is important for meaningful and consistent protein structure analyses. So far there is no widely-used standard for choosing these residue ranges for experimentally determined protein structures, where the manual selection of residue ranges or the use of suboptimal criteria remain commonplace. Results We present an automated and objective method for finding amino acid residue ranges for the superposition and analysis of protein structures, in particular for structure bundles resulting from NMR structure calculations. The method is implemented in an algorithm, CYRANGE, that yields, without protein-specific parameter adjustment, appropriate residue ranges in most commonly occurring situations, including low-precision structure bundles, multi-domain proteins, symmetric multimers, and protein complexes. Residue ranges are chosen to comprise as many residues of a protein domain that increasing their number would lead to a steep rise in the RMSD value. Residue ranges are determined by first clustering residues into domains based on the distance variance matrix, and then refining for each domain the initial choice of residues by excluding residues one by one until the relative decrease of the RMSD value becomes insignificant. A penalty for the opening of gaps favours contiguous residue ranges in order to obtain a result that is as simple as possible, but not simpler. Results are given for a set of 37 proteins and compared with those of commonly used protein structure validation packages. We also provide residue ranges for 6351 NMR structures in the Protein Data Bank. Conclusions The CYRANGE method is capable of automatically determining residue ranges for the superposition of protein structure bundles for a large variety of protein structures. The method correctly identifies ordered regions. Global structure superpositions based on the CYRANGE residue ranges allow a clear presentation of the structure, and unnecessary small gaps within the selected ranges are absent. In the majority of cases, the residue ranges from CYRANGE contain fewer gaps and cover considerably larger parts of the sequence than those from other methods without significantly increasing the RMSD values. CYRANGE thus provides an objective and automatic method for standardizing the choice of residue ranges for the superposition of protein structures. PMID:21592348

The carboxyl tail of connexin32 regulates gap junction assembly in human prostate and pancreatic cancer cells.

PubMed

Katoch, Parul; Mitra, Shalini; Ray, Anuttoma; Kelsey, Linda; Roberts, Brett J; Wahl, James K; Johnson, Keith R; Mehta, Parmender P

2015-02-20

Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A Novel Heat Shock Element (HSE) in Entamoeba histolytica that Regulates the Transcriptional Activation of the EhPgp5 Gene in the Presence of Emetine Drug

PubMed Central

Nieto, Alma; Pérez Ishiwara, David G.; Orozco, Esther; Sánchez Monroy, Virginia; Gómez García, Consuelo

2017-01-01

Transcriptional regulation of the multidrug resistance EhPgp5 gene in Entamoeba histolytica is induced by emetine stress. EhPgp5 overexpression alters the chloride-dependent currents that cause trophozoite swelling, diminishing induced programmed cell death (PCD) susceptibility. In contrast, antisense inhibition of P-glycoprotein (PGP) expression produces synchronous death of trophozoites and the enhancement of the biochemical and morphological characteristics of PCD induced by G418. Transcriptional gene regulation analysis identified a 59 bp region at position −170 to −111 bp promoter as putative emetine response elements (EREs). However, insights into transcription factors controlling EhPgp5 gene transcription are missing; to fill this knowledge gap, we used deletion studies and transient CAT activity assays. Our findings suggested an activating motif (−151 to −136 bp) that corresponds to a heat shock element (HSE). Gel-shift assays, UV-crosslinking, binding protein purification, and western blotting assays revealed proteins of 94, 66, 62, and 51 kDa binding to the EhPgp5 HSE that could be heat shock-like transcription factors that regulate the transcriptional activation of the EhPgp5 gene in the presence of emetine drug. PMID:29238701

The Effects of IGF-1 on TNF-α-Treated DRG Neurons by Modulating ATF3 and GAP-43 Expression via PI3K/Akt/S6K Signaling Pathway.

PubMed

Zhang, Lei; Yue, Yaping; Ouyang, Meishuo; Liu, Huaxiang; Li, Zhenzhong

2017-05-01

Upregulation of the pro-inflammatory cytokine tumor necrosis factor α (TNF-α) is involved in the development and progression of numerous neurological disorders. Recent reports have challenged the concept that TNF-α exhibits only deleterious effects of pro-inflammatory destruction, and have raised the awareness that it may play a beneficial role in neuronal growth and function in particular conditions, which prompts us to further investigate the role of this cytokine. Insulin-like growth factor-1 (IGF-1) is a cytokine possessing powerful neuroprotective effects in promoting neuronal survival, neuronal differentiation, neurite elongation, and neurite regeneration. The association of IGF-1 with TNF-α and the biological effects, produced by interaction of IGF-1 and TNF-α, on neuronal outgrowth status of primary sensory neurons are still to be clarified. In the present study, using an in vitro model of primary cultured rat dorsal root ganglion (DRG) neurons, we demonstrated that TNF-α challenge at different concentrations elicited diverse biological effects. Higher concentration of TNF-α (10 ng/mL) dampened neurite outgrowth, induced activating transcription factor 3 (ATF3) expression, reduced growth-associated protein 43 (GAP-43) expression, and promoted GAP-43 and ATF3 coexpression, which could be reversed by IGF-1 treatment; while lower concentration of TNF-α (1 ng/mL) promoted neurite sprouting, decreased ATF3 expression, increased GAP-43 expression, and inhibited GAP-43 and ATF3 coexpression, which could be potentiated by IGF-1 supplement. Moreover, IGF-1 administration restored the activation of Akt and p70 S6 kinase (S6K) suppressed by higher concentration of TNF-α (10 ng/mL) challenge. In contrast, lower concentration of TNF-α (1 ng/mL) had no significant effect on Akt or S6K activation, and IGF-1 administration activated these two kinases. The effects of IGF-1 were abrogated by phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. These data imply that IGF-1 counteracts the toxic effect of higher concentration of TNF-α, while potentiates the growth-promoting effect of lower concentration of TNF-α, with the node for TNF-α and IGF-1 interaction being the PI3K/Akt/S6K signaling pathway. This study is helpful for interpretation of the association of IGF-1 with TNF-α and the neurobiological effects elicited by interaction of IGF-1 and TNF-α in neurological disorders.

Amygdala stimulation promotes recovery of behavioral performance in a spatial memory task and increases GAP-43 and MAP-2 in the hippocampus and prefrontal cortex of male rats.

PubMed

Mercerón-Martínez, D; Almaguer-Melian, W; Alberti-Amador, E; Bergado, J A

2018-06-19

The relationships between affective and cognitive processes are an important issue of present neuroscience. The amygdala, the hippocampus and the prefrontal cortex appear as main players in these mechanisms. We have shown that post-training electrical stimulation of the basolateral amygdala (BLA) speeds the acquisition of a motor skill, and produces a recovery in behavioral performance related to spatial memory in fimbria-fornix (FF) lesioned animals. BLA electrical stimulation rises bdnf RNA expression, BDNF protein levels, and arc RNA expression in the hippocampus. In the present paper we have measured the levels of one presynaptic protein (GAP-43) and one postsynaptic protein (MAP-2) both involved in synaptogenesis to assess whether structural neuroplastic mechanisms are involved in the memory enhancing effects of BLA stimulation. A single train of BLA stimulation produced in healthy animals an increase in the levels of GAP-43 and MAP-2 that lasted days in the hippocampus and the prefrontal cortex. In FF-lesioned rats, daily post-training stimulation of the BLA ameliorates the memory deficit of the animals and induces an increase in the level of both proteins. These results support the hypothesis that the effects of amygdala stimulation on memory recovery are sustained by an enhanced formation of new synapses. Copyright © 2018. Published by Elsevier Inc.

Regulation of blood-testis barrier dynamics by desmosome, gap junction, hemidesmosome and polarity proteins

PubMed Central

Wong, Elissa WP; Lie, Pearl PY; Li, Michelle WM; Mruk, Dolores D; Yan, Helen HN; Mok, Ka-Wai; Mannu, Jayakanthan; Mathur, Premendu P; Lui, Wing-yee; Lee, Will M; Bonanomi, Michele; Silvestrini, Bruno

2011-01-01

The blood-testis barrier (BTB) is a unique ultrastructure in the mammalian testis. Unlike other blood-tissue barriers, such as the blood-brain barrier and the blood-ocular (or blood-retina) barrier which formed by tight junctions (TJ) between endothelial cells of the microvessels, the BTB is constituted by coexisting TJ, basal ectoplasmic specialization (basal ES), desmosomes and gap junctions between adjacent Sertoli cells near the basement membrane of the seminiferous tubule. The BTB also divides the seminiferous epithelium into the apical (or adluminal) and basal compartments so that meiosis I and II and post-meiotic germ cell development can all take place in a specialized microenvironment in the apical compartment behind the BTB. While the unusual anatomical features of the BTB have been known for decades, the physiological function of the coexisting junctions, in particular the desmosome and gap junction, that constitute the BTB was unknown until recently. Based on recently published findings, we critically evaluate the role of the desmosome and gap junction that serve as a signaling platform to coordinate the “opening” and “closing” of the TJ-permeability barrier conferred by TJ and basal ES during the seminiferous epithelial cycle of spermatogenesis. This is made possible by polarity proteins working in concert with nonreceptor protein tyrosine kinases, such as focal adhesion kinase (FAK) and c-Src, at the site to regulate endosome-mediated protein trafficking events (e.g., endocytosis, transcytosis, recycling or protein degradation). These events not only serve to destabilize the existing “old” BTB above preleptotene spermatocytes in transit in “clones” at the BTB, but also contribute to the assembly of “new” BTB below the transiting spermatocytes. Furthermore, hemidesmosomes at the Sertoli cell-basement membrane interface also contribute to the BTB restructuring events at stage VIII of the epithelial cycle. Additionally, the findings that a gap junction at the BTB provides a possible route for the passage of toxicants [e.g., bisphenol A (BPA)] and potential male contraceptives (e.g., adjudin) across the BTB also illustrate that these coexisting junctions, while helpful to maintain the immunological barrier integrity during the transit of spermatocytes, can be the “gateway” to making the BTB so vulnerable to toxicants and/or chemicals, causing male reproductive dysfunction. PMID:22319658

Adaptive Evolution of Signaling Partners

PubMed Central

Urano, Daisuke; Dong, Taoran; Bennetzen, Jeffrey L.; Jones, Alan M.

2015-01-01

Proteins that interact coevolve their structures. When mutation disrupts the interaction, compensation by the partner occurs to restore interaction otherwise counterselection occurs. We show in this study how a destabilizing mutation in one protein is compensated by a stabilizing mutation in its protein partner and their coevolving path. The pathway in this case and likely a general principle of coevolution is that the compensatory change must tolerate both the original and derived structures with equivalence in function and activity. Evolution of the structure of signaling elements in a network is constrained by specific protein pair interactions, by requisite conformational changes, and by catalytic activity. The heterotrimeric G protein-coupled signaling is a paragon of this protein interaction/function complexity and our deep understanding of this pathway in diverse organisms lends itself to evolutionary study. Regulators of G protein Signaling (RGS) proteins accelerate the intrinsic GTP hydrolysis rate of the Gα subunit of the heterotrimeric G protein complex. An important RGS-contact site is a hydroxyl-bearing residue on the switch I region of Gα subunits in animals and most plants, such as Arabidopsis. The exception is the grasses (e.g., rice, maize, sugarcane, millets); these plants have Gα subunits that replaced the critical hydroxyl-bearing threonine with a destabilizing asparagine shown to disrupt interaction between Arabidopsis RGS protein (AtRGS1) and the grass Gα subunit. With one known exception (Setaria italica), grasses do not encode RGS genes. One parsimonious deduction is that the RGS gene was lost in the ancestor to the grasses and then recently acquired horizontally in the lineage S. italica from a nongrass monocot. Like all investigated grasses, S. italica has the Gα subunit with the destabilizing asparagine residue in the protein interface but, unlike other known grass genomes, still encodes an expressed RGS gene, SiRGS1. SiRGS1 accelerates GTP hydrolysis at similar concentration of both Gα subunits containing either the stabilizing (AtGPA1) or destabilizing (RGA1) interface residue. SiRGS1 does not use the hydroxyl-bearing residue on Gα to promote GAP activity and has a larger Gα-interface pocket fitting to the destabilizing Gα. These findings indicate that SiRGS1 adapted to a deleterious mutation on Gα using existing polymorphism in the RGS protein population. PMID:25568345

Monocytic cell junction proteins serve important roles in atherosclerosis via the endoglin pathway

PubMed Central

Chen, Lina; Chen, Zhongliang; Ge, Menghua; Tang, Oushan; Cheng, Yinhong; Zhou, Haoliang; Shen, Yu; Qin, Fengming

2017-01-01

The formation of atherosclerosis is recognized to be caused by multiple factors including pathogenesis in monocytes during inflammation. The current study provided evidence that monocytic junctions were significantly altered in patients with atherosclerosis, which suggested an association between cell junctions and atherosclerosis. Claudin-1, occludin-1 and ZO-1 were significantly enhanced in atherosclerosis, indicating that the tight junction pathway was activated during the pathogenesis of atherosclerosis. In addition, the gene expression of 5 connexin members involved in the gap junction pathway were quantified, indicating that connexin 43 and 46 were significantly up-regulated in atherosclerosis. Furthermore, inflammatory factors including endoglin and SMAD were observed, suggesting that immune regulative factors were down-regulated in this pathway. Silicon-based analysis additionally identified that connexins and tight junctions were altered in association with monocytic inflammation regulations, endoglin pathway. The results imply that reduced expression of the immune regulation pathway in monocytes is correlated with the generation of gap junctions and tight junctions which serve important roles in atherosclerosis. PMID:28901429

Reduced insulin signaling maintains electrical transmission in a neural circuit in aging flies

PubMed Central

McGourty, Kieran; Allen, Marcus J.; Madem, Sirisha Kudumala; Adcott, Jennifer; Kerr, Fiona; Wong, Chi Tung; Vincent, Alec; Godenschwege, Tanja; Boucrot, Emmanuel; Partridge, Linda

2017-01-01

Lowered insulin/insulin-like growth factor (IGF) signaling (IIS) can extend healthy lifespan in worms, flies, and mice, but it can also have adverse effects (the “insulin paradox”). Chronic, moderately lowered IIS rescues age-related decline in neurotransmission through the Drosophila giant fiber system (GFS), a simple escape response neuronal circuit, by increasing targeting of the gap junctional protein innexin shaking-B to gap junctions (GJs). Endosomal recycling of GJs was also stimulated in cultured human cells when IIS was reduced. Furthermore, increasing the activity of the recycling small guanosine triphosphatases (GTPases) Rab4 or Rab11 was sufficient to maintain GJs upon elevated IIS in cultured human cells and in flies, and to rescue age-related loss of GJs and of GFS function. Lowered IIS thus elevates endosomal recycling of GJs in neurons and other cell types, pointing to a cellular mechanism for therapeutic intervention into aging-related neuronal disorders. PMID:28902870

Aberrant activity in retinal degeneration impairs central visual processing and relies on Cx36-containing gap junctions.

PubMed

Ivanova, Elena; Yee, Christopher W; Baldoni, Robert; Sagdullaev, Botir T

2016-09-01

In retinal degenerative disease (RD), the diminished light signal from dying photoreceptors has been considered the sole cause of visual impairment. Recent studies show a 10-fold increase in spontaneous activity in the RD network, challenging this paradigm. This aberrant activity forms a new barrier for the light signal, and not only exacerbates the loss of vision, but also may stand in the way of visual restoration. This activity originates in AII amacrine cells and relies on excessive activation of gap junctions. However, it remains unclear whether aberrant activity affects central visual processing and what mechanisms lead to this excessive activation of gap junctions. By combining genetic manipulation with electrophysiological recordings of light-induced activity in both living mice and isolated wholemount retina, we demonstrate that aberrant activity extends along retinotectal projections to alter activity in higher brain centers. Next, to selectively eliminate Cx36-containing gap junctions, which are the primary type expressed by AII amacrine cells, we crossed rd10 mice, a slow-degenerating model of RD, with Cx36 knockout mice. We found that retinal aberrant activity was reduced in the rd10/Cx36KO mice compared to rd10 controls, a direct evidence for involvement of Cx36-containing gap junctions in generating aberrant activity in RD. These data provide an essential support for future experiments to determine if selectively targeting these gap junctions could be a valid strategy for reducing aberrant activity and restoring light responses in RD. Copyright © 2015 Elsevier Ltd. All rights reserved.

A tip-localized RhoGAP controls cell polarity by globally inhibiting Rho GTPase at the cell apex.

PubMed

Hwang, Jae-Ung; Vernoud, Vanessa; Szumlanski, Amy; Nielsen, Erik; Yang, Zhenbiao

2008-12-23

Highly elongated eukaryotic cells (e.g., neuronal axons, fungal hyphae, and pollen tubes) are generated through continuous apically restricted growth (tip growth), which universally requires tip-localized Rho GTPases. We used the oscillating pollen tube as a model system to determine the function and regulation of Rho GTPases in tip growth. Our previous work showed that the spatiotemporal dynamics of the apical cap of the activated Rho-like GTPase from Plant 1 (ROP1) are critical for tip growth in pollen tubes. However, the underlying mechanism for the generation and maintenance of this dynamic apical cap is poorly understood. A screen for mutations that enhance ROP1-overexpression-induced depolarization of pollen-tube growth identified REN1 (ROP1 enhancer 1) in Arabidopsis, whose null mutations turn elongated pollen tubes into bulbous cells. REN1 encodes a novel Rho GTPase-activating protein (RhoGAP) required for restricting the ROP1 activity to the pollen-tube tip. REN1 was localized to exocytic vesicles accumulated in the pollen-tube apex, as well as to the apical plasma membrane at the site of ROP1 activation. The apical localization of REN1 and its function in controlling growth polarity was compromised by disruption of ROP1-dependent F-actin and vesicular trafficking, which indicates that REN1 targeting and function is regulated by ROP1 downstream signaling. Our findings suggest that the REN1 RhoGAP controls a negative-feedback-based global inhibition of ROP1. This function provides a critical self-organizing mechanism, by which ROP signaling is spatially limited to the growth site and temporally oscillates during continuous tip growth. Similar spatiotemporal control of Rho GTPase signaling may also play an important role in cell-polarity control in other systems, including tip growth in fungi and cell movement in animals.

Coiled Coil Rich Proteins (Ccrp) Influence Molecular Pathogenicity of Helicobacter pylori

PubMed Central

Schätzle, Sarah; Specht, Mara; Waidner, Barbara

2015-01-01

Pathogenicity of the human pathogen Helicobacter pylori relies on its capacity to adapt to a hostile environment and to escape the host response. Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its contribution to virulence. In this study we have explored the influence of coiled coil rich proteins (Ccrp) cytoskeletal elements on pathogenicity factors of H. pylori. Deletion of any of the ccrp resulted in a strongly decreased activity of the main pathogenicity factor urease. We further investigated their role using in vitro co-culture experiments with the human gastric adenocarcinoma cell line AGS modeling H. pylori - host cell interactions. Intriguingly, host cell showed only a weak “scattering/hummingbird” phenotype, in which host cells are transformed from a uniform polygonal shape into a severely elongated state characterized by the formation of needle-like projections, after co-incubation with any ccrp deletion mutant. Furthermore, co-incubation with the ccrp59 mutant resulted in reduced type IV secretion system associated activities, e.g. IL-8 production and CagA translocation/phosphorylation. Thus, in addition to their role in maintaining the helical cell shape of H. pylori Ccrp proteins influence many cellular processes and are thereby crucial for the virulence of this human pathogen. PMID:25822999

Pado, a fluorescent protein with proton channel activity can optically monitor membrane potential, intracellular pH, and map gap junctions.

PubMed

Kang, Bok Eum; Baker, Bradley J

2016-04-04

An in silico search strategy was developed to identify potential voltage-sensing domains (VSD) for the development of genetically encoded voltage indicators (GEVIs). Using a conserved charge distribution in the S2 α-helix, a single in silico search yielded most voltage-sensing proteins including voltage-gated potassium channels, voltage-gated calcium channels, voltage-gated sodium channels, voltage-gated proton channels, and voltage-sensing phosphatases from organisms ranging from mammals to bacteria and plants. A GEVI utilizing the VSD from a voltage-gated proton channel identified from that search was able to optically report changes in membrane potential. In addition this sensor was capable of manipulating the internal pH while simultaneously reporting that change optically since it maintains the voltage-gated proton channel activity of the VSD. Biophysical characterization of this GEVI, Pado, demonstrated that the voltage-dependent signal was distinct from the pH-dependent signal and was dependent on the movement of the S4 α-helix. Further investigation into the mechanism of the voltage-dependent optical signal revealed that inhibiting the dimerization of the fluorescent protein greatly reduced the optical signal. Dimerization of the FP thereby enabled the movement of the S4 α-helix to mediate a fluorescent response.

Pado, a fluorescent protein with proton channel activity can optically monitor membrane potential, intracellular pH, and map gap junctions

PubMed Central

Kang, Bok Eum; Baker, Bradley J.

2016-01-01

An in silico search strategy was developed to identify potential voltage-sensing domains (VSD) for the development of genetically encoded voltage indicators (GEVIs). Using a conserved charge distribution in the S2 α-helix, a single in silico search yielded most voltage-sensing proteins including voltage-gated potassium channels, voltage-gated calcium channels, voltage-gated sodium channels, voltage-gated proton channels, and voltage-sensing phosphatases from organisms ranging from mammals to bacteria and plants. A GEVI utilizing the VSD from a voltage-gated proton channel identified from that search was able to optically report changes in membrane potential. In addition this sensor was capable of manipulating the internal pH while simultaneously reporting that change optically since it maintains the voltage-gated proton channel activity of the VSD. Biophysical characterization of this GEVI, Pado, demonstrated that the voltage-dependent signal was distinct from the pH-dependent signal and was dependent on the movement of the S4 α-helix. Further investigation into the mechanism of the voltage-dependent optical signal revealed that inhibiting the dimerization of the fluorescent protein greatly reduced the optical signal. Dimerization of the FP thereby enabled the movement of the S4 α-helix to mediate a fluorescent response. PMID:27040905

Thalamic modulation of cingulate seizure activity via the regulation of gap junctions in mice thalamocingulate slice.

PubMed

Chang, Wei-Pang; Wu, José Jiun-Shian; Shyu, Bai-Chuang

2013-01-01

The thalamus is an important target for deep brain stimulation in the treatment of seizures. However, whether the modulatory effect of thalamic inputs on cortical seizures occurs through the modulation of gap junctions has not been previously studied. Therefore, we tested the effects of different gap junction blockers and couplers in a drug-resistant seizure model and studied the role of gap junctions in the thalamic modulation on cortical seizures. Multielectrode array and calcium imaging were used to record the cortical seizures induced by 4-aminopyridine (250 µM) and bicuculline (5-50 µM) in a novel thalamocingulate slice preparation. Seizure-like activity was significantly attenuated by the pan-gap junction blockers carbenoxolone and octanol and specific neuronal gap junction blocker mefloquine. The gap junction coupler trimethylamine significantly enhanced seizure-like activity. Gap junction blockers did not influence the initial phase of seizure-like activity, but they significantly decreased the amplitude and duration of the maintenance phase. The development of seizures is regulated by extracellular potassium concentration. Carbenoxolone partially restored the amplitude and duration after removing the thalamic inputs. A two-dimensional current source density analysis showed that the sink and source signals shifted to deeper layers after removing the thalamic inputs during the clonic phase. These results indicate that the regulatory mechanism of deep brain stimulation in the thalamus occurs partially though gap junctions.

Thalamic Modulation of Cingulate Seizure Activity Via the Regulation of Gap Junctions in Mice Thalamocingulate Slice

PubMed Central

Chang, Wei-Pang; Wu, José Jiun-Shian; Shyu, Bai-Chuang

2013-01-01

The thalamus is an important target for deep brain stimulation in the treatment of seizures. However, whether the modulatory effect of thalamic inputs on cortical seizures occurs through the modulation of gap junctions has not been previously studied. Therefore, we tested the effects of different gap junction blockers and couplers in a drug-resistant seizure model and studied the role of gap junctions in the thalamic modulation on cortical seizures. Multielectrode array and calcium imaging were used to record the cortical seizures induced by 4-aminopyridine (250 µM) and bicuculline (5–50 µM) in a novel thalamocingulate slice preparation. Seizure-like activity was significantly attenuated by the pan-gap junction blockers carbenoxolone and octanol and specific neuronal gap junction blocker mefloquine. The gap junction coupler trimethylamine significantly enhanced seizure-like activity. Gap junction blockers did not influence the initial phase of seizure-like activity, but they significantly decreased the amplitude and duration of the maintenance phase. The development of seizures is regulated by extracellular potassium concentration. Carbenoxolone partially restored the amplitude and duration after removing the thalamic inputs. A two-dimensional current source density analysis showed that the sink and source signals shifted to deeper layers after removing the thalamic inputs during the clonic phase. These results indicate that the regulatory mechanism of deep brain stimulation in the thalamus occurs partially though gap junctions. PMID:23690968

Bridging the gap between protein carboxyl methylation and phospholipid methylation to understand glucose-stimulated insulin secretion from the pancreatic beta cell.

PubMed

Kowluru, Anjaneyulu

2008-01-15

Recent findings have implicated post-translational modifications at C-terminal cysteines [e.g., methylation] of specific proteins [e.g., G-proteins] in glucose-stimulated insulin secretion [GSIS]. Furthermore, methylation at the C-terminal leucine of the catalytic subunit of protein phosphatase 2A [PP2Ac] has also been shown to be relevant for GSIS. In addition to these two classes of protein methyl transferases, a novel class of glucose-activated phospholipid methyl transferases have also been identified in the beta cell. These enzymes catalyze three successive methylations of phosphatidylethanolamine to yield phosphatidylcholine. The "newly formed" phosphatidylcholine is felt to induce alterations in the membrane fluidity, which might favor vesicular fusion with the plasma membrane for the exocytosis of insulin. The objectives of this commentary are to: (i) review the existing evidence on the regulation, by glucose and other insulin secretagogues, of post-translational carboxylmethylation [CML] of specific proteins in the beta cell; (ii) discuss the experimental evidence, which implicates regulation, by glucose and other insulin secretagogues, of phosphatidylethanolamine methylation in the islet beta cell; (iii) propose a model for potential cross-talk between the protein and lipid methylation pathways in the regulation of GSIS and (iv) highlight potential avenues for future research, including the development of specific pharmacological inhibitors to further decipher regulatory roles for these methylation reactions in islet beta cell function.

Toward Bridging the Mechanistic Gap between Genes and Traits by Emphasizing the Role of Proteins in a Computational Environment

ERIC Educational Resources Information Center

Haskel-Ittah, Michal; Yarden, Anat

2017-01-01

Previous studies have shown that students often ignore molecular mechanisms when describing genetic phenomena. Specifically, students tend to directly link genes to their encoded traits, ignoring the role of proteins as mediators in this process. We tested the ability of 10th grade students to connect genes to traits through proteins, using…

Gap Junctions Contribute to the Regulation of Walking-Like Activity in the Adult Mudpuppy (Necturus Maculatus).

PubMed

Lavrov, Igor; Fox, Lyle; Shen, Jun; Han, Yingchun; Cheng, Jianguo

2016-01-01

Although gap junctions are widely expressed in the developing central nervous system, the role of electrical coupling of neurons and glial cells via gap junctions in the spinal cord in adults is largely unknown. We investigated whether gap junctions are expressed in the mature spinal cord of the mudpuppy and tested the effects of applying gap junction blocker on the walking-like activity induced by NMDA or glutamate in an in vitro mudpuppy preparation. We found that glial and neural cells in the mudpuppy spinal cord expressed different types of connexins that include connexin 32 (Cx32), connexin 36 (Cx36), connexin 37 (Cx37), and connexin 43 (Cx43). Application of a battery of gap junction blockers from three different structural classes (carbenexolone, flufenamic acid, and long chain alcohols) substantially and consistently altered the locomotor-like activity in a dose-dependent manner. In contrast, these blockers did not significantly change the amplitude of the dorsal root reflex, indicating that gap junction blockers did not inhibit neuronal excitability nonselectively in the spinal cord. Taken together, these results suggest that gap junctions play a significant modulatory role in the spinal neural networks responsible for the generation of walking-like activity in the adult mudpuppy.

Dietary protein-induced hepatic IGF-1 secretion mediated by PPARγ activation.

PubMed

Wan, Xiaojuan; Wang, Songbo; Xu, Jingren; Zhuang, Lu; Xing, Kongping; Zhang, Mengyuan; Zhu, Xiaotong; Wang, Lina; Gao, Ping; Xi, Qianyun; Sun, Jiajie; Zhang, Yongliang; Li, Tiejun; Shu, Gang; Jiang, Qingyan

2017-01-01

Dietary protein or amino acid (AA) is a crucial nutritional factor to regulate hepatic insulin-like growth factor-1 (IGF-1) expression and secretion. However, the underlying intracellular mechanism by which dietary protein or AA induces IGF-1 expression remains unknown. We compared the IGF-1 gene expression and plasma IGF-1 level of pigs fed with normal crude protein (CP, 20%) and low-protein levels (LP, 14%). RNA sequencing (RNA-seq) was performed to detect transcript expression in the liver in response to dietary protein. The results showed that serum concentrations and mRNA levels of IGF-1 in the liver were higher in the CP group than in the LP group. RNA-seq analysis identified a total of 1319 differentially expressed transcripts (667 upregulated and 652 downregulated), among which the terms "oxidative phosphorylation", "ribosome", "gap junction", "PPAR signaling pathway", and "focal adhesion" were enriched. In addition, the porcine primary hepatocyte and HepG2 cell models also demonstrated that the mRNA and protein levels of IGF-1 and PPARγ increased with the increasing AA concentration in the culture. The PPARγ activator troglitazone increased IGF-1 gene expression and secretion in a dose dependent manner. Furthermore, inhibition of PPARγ effectively reversed the effects of the high AA concentration on the mRNA expression of IGF-1 and IGFBP-1 in HepG2 cells. Moreover, the protein levels of IGF-1 and PPARγ, as well as the phosphorylation of mTOR, significantly increased in HepG2 cells under high AA concentrations. mTOR phosphorylation can be decreased by the mTOR antagonist, rapamycin. The immunoprecipitation results also showed that high AA concentrations significantly increased the interaction of mTOR and PPARγ. In summary, PPARγ plays an important role in the regulation of IGF-1 secretion and gene expression in response to dietary protein.

Cell cycle-dependent Rho GTPase activity dynamically regulates cancer cell motility and invasion in vivo.

PubMed

Kagawa, Yoshinori; Matsumoto, Shinji; Kamioka, Yuji; Mimori, Koshi; Naito, Yoko; Ishii, Taeko; Okuzaki, Daisuke; Nishida, Naohiro; Maeda, Sakae; Naito, Atsushi; Kikuta, Junichi; Nishikawa, Keizo; Nishimura, Junichi; Haraguchi, Naotsugu; Takemasa, Ichiro; Mizushima, Tsunekazu; Ikeda, Masataka; Yamamoto, Hirofumi; Sekimoto, Mitsugu; Ishii, Hideshi; Doki, Yuichiro; Matsuda, Michiyuki; Kikuchi, Akira; Mori, Masaki; Ishii, Masaru

2013-01-01

The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci) demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP), was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers.

Cell Cycle-Dependent Rho GTPase Activity Dynamically Regulates Cancer Cell Motility and Invasion In Vivo

PubMed Central

Kagawa, Yoshinori; Matsumoto, Shinji; Kamioka, Yuji; Mimori, Koshi; Naito, Yoko; Ishii, Taeko; Okuzaki, Daisuke; Nishida, Naohiro; Maeda, Sakae; Naito, Atsushi; Kikuta, Junichi; Nishikawa, Keizo; Nishimura, Junichi; Haraguchi, Naotsugu; Takemasa, Ichiro; Mizushima, Tsunekazu; Ikeda, Masataka; Yamamoto, Hirofumi; Sekimoto, Mitsugu; Ishii, Hideshi; Doki, Yuichiro; Matsuda, Michiyuki; Kikuchi, Akira; Mori, Masaki; Ishii, Masaru

2013-01-01

The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci) demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP), was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers. PMID:24386239

Course 12: Proteins: Structural, Thermodynamic and Kinetic Aspects

NASA Astrophysics Data System (ADS)

Finkelstein, A. V.

1 Introduction 2 Overview of protein architectures and discussion of physical background of their natural selection 2.1 Protein structures 2.2 Physical selection of protein structures 3 Thermodynamic aspects of protein folding 3.1 Reversible denaturation of protein structures 3.2 What do denatured proteins look like? 3.3 Why denaturation of a globular protein is the first-order phase transition 3.4 "Gap" in energy spectrum: The main characteristic that distinguishes protein chains from random polymers 4 Kinetic aspects of protein folding 4.1 Protein folding in vivo 4.2 Protein folding in vitro (in the test-tube) 4.3 Theory of protein folding rates and solution of the Levinthal paradox

Role of gap junctions in the contractile response to agonists in the mesenteric artery of spontaneously hypertensive rats.

PubMed

Ma, Ke-Tao; Li, Xin-Zhi; Li, Li; Jiang, Xue-Wei; Chen, Xin-Yan; Liu, Wei-Dong; Zhao, Lei; Zhang, Zhong-Shuang; Si, Jun-Qiang

2014-02-01

To investigate the effects of hypertension on the changes in gap junctions between vascular smooth muscle cells (VSMCs) in the mesenteric artery (MA) of spontaneously hypertensive rats (SHRs). Whole-cell patch clamp, pressure myography, real-time quantitative reverse transcription PCR (qRT-PCR), western blot analysis and transmission electron microscopy were used to examine the differences in expression and function of the gap junction between MA VSMCs of SHR and control normotensive Wistar-Kyoto (WKY) rats. (1) Whole-cell patch clamp measurements showed that the membrane capacitance and conductance of in-situ MA VSMCs of SHR were significantly greater than those of WKY rats (P<0.05), suggesting enhanced gap junction coupling between MA VSMCs of SHR. (2) The administration of phenylephrine (PE) and KCl (an endothelium-independent vasoconstrictor) initiated more pronounced vasoconstriction in SHR versus WKY rats (P<0.05). Furthermore, 2-APB (a gap junction inhibitor) attenuated PE- and KCl-induced vasoconstriction, and the inhibitory effects of 2-APB were significantly greater in SHR (P<0.05). (3) The expression of connexin 45 (Cx45) mRNA and protein in the MA was greater in SHR versus WKY rats (P<0.05). The level of phosphorylated Cx43 was significantly higher in SHR versus WKY rats (P<0.05), although the expression of total Cx43 mRNA and protein in the MA was equivalent between SHR and WKY rats. Electron microscopy revealed that the gap junctions were significantly larger in SHR versus WKY rats. Increases in the expression of Cx45 and phosphorylation of Cx43 may contribute to the enhancement of communication across gap junctions between MA VSMCs of SHR, which may increase the contractile response to agonists.

KSRP Modulation of GAP-43 mRNA Stability Restricts Axonal Outgrowth in Embryonic Hippocampal Neurons

PubMed Central

Bird, Clark W.; Gardiner, Amy S.; Bolognani, Federico; Tanner, Daniel C.; Chen, Ching-Yi; Lin, Wei-Jye; Yoo, Soonmoon; Twiss, Jeffery L.; Perrone- Bizzozero, Nora

2013-01-01

The KH-type splicing regulatory protein (KSRP) promotes the decay of AU-rich element (ARE)-containing mRNAs. Although KSRP is expressed in the nervous system, very little is known about its role in neurons. In this study, we examined whether KSRP regulates the stability of the ARE-containing GAP-43 mRNA. We found that KSRP destabilizes this mRNA by binding to its ARE, a process that requires the presence of its fourth KH domain (KH4). Furthermore, KSRP competed with the stabilizing factor HuD for binding to these sequences. We also examined the functional consequences of KSRP overexpression and knockdown on the differentiation of primary hippocampal neurons in culture. Overexpression of full length KSRP or KSRP without its nuclear localization signal hindered axonal outgrowth in these cultures, while overexpression of a mutant protein without the KH4 domain that has less affinity for binding to GAP-43′s ARE had no effect. In contrast, depletion of KSRP led to a rise in GAP-43 mRNA levels and a dramatic increase in axonal length, both in KSRP shRNA transfected cells and neurons cultured from Ksrp+/− and Ksrp −/−embryos. Finally we found that overexpression of GAP-43 rescued the axonal outgrowth deficits seen with KSRP overexpression, but only when cells were transfected with GAP-43 constructs containing 3′ UTR sequences targeting the transport of this mRNA to axons. Together, our results suggest that KSRP is an important regulator of mRNA stability and axonal length that works in direct opposition to HuD to regulate the levels of GAP-43 and other ARE-containing neuronal mRNAs. PMID:24244461

Gap junctional coupling in the vertebrate retina: variations on one theme?

PubMed

Völgyi, Béla; Kovács-Oller, Tamás; Atlasz, Tamás; Wilhelm, Márta; Gábriel, Róbert

2013-05-01

Gap junctions connect cells in the bodies of all multicellular organisms, forming either homologous or heterologous (i.e. established between identical or different cell types, respectively) cell-to-cell contacts by utilizing identical (homotypic) or different (heterotypic) connexin protein subunits. Gap junctions in the nervous system serve electrical signaling between neurons, thus they are also called electrical synapses. Such electrical synapses are particularly abundant in the vertebrate retina where they are specialized to form links between neurons as well as glial cells. In this article, we summarize recent findings on retinal cell-to-cell coupling in different vertebrates and identify general features in the light of the evergrowing body of data. In particular, we describe and discuss tracer coupling patterns, connexin proteins, junctional conductances and modulatory processes. This multispecies comparison serves to point out that most features are remarkably conserved across the vertebrate classes, including (i) the cell types connected via electrical synapses; (ii) the connexin makeup and the conductance of each cell-to-cell contact; (iii) the probable function of each gap junction in retinal circuitry; (iv) the fact that gap junctions underlie both electrical and/or tracer coupling between glial cells. These pan-vertebrate features thus demonstrate that retinal gap junctions have changed little during the over 500 million years of vertebrate evolution. Therefore, the fundamental architecture of electrically coupled retinal circuits seems as old as the retina itself, indicating that gap junctions deeply incorporated in retinal wiring from the very beginning of the eye formation of vertebrates. In addition to hard wiring provided by fast synaptic transmitter-releasing neurons and soft wiring contributed by peptidergic, aminergic and purinergic systems, electrical coupling may serve as the 'skeleton' of lateral processing, enabling important functions such as signal averaging and synchronization. 2013 Elsevier Ltd. All rights reserved.