scholarly journals Role for Gcs1p in Regulation of Arl1p atTrans-Golgi Compartments

2005 ◽  
Vol 16 (9) ◽  
pp. 4024-4033 ◽  
Author(s):  
Ya-Wen Liu ◽  
Chun-Fang Huang ◽  
Kai-Bin Huang ◽  
Fang-Jen S. Lee
Keyword(s):  
Dynamic Equilibrium ◽  
Vesicular Trafficking ◽  
Dependent Manner ◽  
Wild Type ◽  
Gtpase Activating Protein ◽  
Critical Components ◽  
Mutant Cells ◽  
Hydrolysis Of ◽  
Adp Ribosylation Factor

ADP-ribosylation factor (ARF) and ARF-like (ARL) proteins are members of the ARF family, which are critical components of several different vesicular trafficking pathways. ARFs have little or no detectable GTPase activity without the assistance of a GTPase-activating protein (GAP). Here, we demonstrate that yeast Gcs1p exhibits GAP activity toward Arl1p and Arf1p in vitro, and Arl1p can interact with Gcs1p in a GTP-dependent manner. Arl1p was observed both on trans-Golgi and in cytosol and was recruited from cytosol to membranes in a GTP-dependent manner. In gcs1 mutant cells, the fraction of Arl1p in cytosol relative to trans-Golgi was less than it was in wild-type cells. Increasing Gcs1p levels returned the distribution toward that of wild-type cells. Both Arl1p and Gcs1p influenced the distribution of Imh1p, an Arl1p effector. Our data are consistent with the conclusion that Arl1p moves in a dynamic equilibrium between trans-Golgi and cytosol, and the release of Arl1p from membranes in cells requires the hydrolysis of bound GTP, which is accelerated by Gcs1p.

scholarly journals A Common Variant of ASAP1 Is Associated with Tuberculosis Susceptibility in the Han Chinese Population

2019 ◽  
Vol 2019 ◽  
pp. 1-7 ◽  
Author(s):  
Cheng Chen ◽  
Qi Zhao ◽  
Yan Shao ◽  
Yan Li ◽  
Honghuan Song ◽  
...  
Keyword(s):  
Genetic Variant ◽  
Peripheral Blood Monocyte ◽  
Gtpase Activating Protein ◽  
Potential Biomarker ◽  
Significant Locus ◽  
Latent Tb ◽  
Hydrolysis Of ◽  
Adp Ribosylation Factor ◽  
Rna Fold

Background. ASAP1 (also known as AMAP1 or DDEF1) encodes an Arf GTPase-activating protein (Arf GAP), a multifunctional scaffold protein that induces hydrolysis of GTP bound to the ADP-ribosylation factor (Arf) family GTP-binding proteins. Reduction of ASAP1 expression in vitro was related to suppression of cell migration and invasiveness. The genetic variant rs4733781 of the ASAP1 gene was revealed as a significant locus for tuberculosis (TB) susceptibility, but the results still need to be validated. Methods. Blood samples from a total of 1914 active TB and healthy controls (HC) were collected to evaluate rs4733781 and the risk of TB. Meanwhile, a total of 48 noninfected HC, latent TB-infected (LTBI) controls, and active TB were collected to assay ASAP1 expression difference among the three groups. The QuantiFERON-TB Gold In-Tube was adopted to identify noninfected HC and LTBI. Results. The genetic variant of rs4733781 was found to be significantly associated with TB, and the A allele of rs4733781 (C>A) was 0.38 and 0.43 among TB cases and HC, respectively (P=0.0035). Meanwhile, the peripheral blood monocyte RNA fold changes for the ASAP1 gene among the 16 HC, 16 LTBI, and 16 active TB were 1.088±0.4919, 2.237±0.6505, and 10.12±10.98 (F=9.559, P=0.0003), respectively, and the expression of ASAP1 was increased by 2.06-fold (P<0.0001) and 9.30-fold (P<0.0052) for LTBI and active TB, when compared to the HC. Conclusions. Our data indicated that the A allele of rs4733781 for the ASAP1 gene was in association with a decreased risk of TB. But not only that, the overexpression of the ASAP1 gene among LTBI and TB was related to the progression of TB, which further implies that the expression of ASAP1 would be a potential biomarker for LTBI and TB diagnoses.

scholarly journals ASAP1, a Phospholipid-Dependent Arf GTPase-Activating Protein That Associates with and Is Phosphorylated by Src

1998 ◽  
Vol 18 (12) ◽  
pp. 7038-7051 ◽  
Author(s):  
Megan T. Brown ◽  
Josefa Andrade ◽  
Harish Radhakrishna ◽  
Julie G. Donaldson ◽  
Jonathan A. Cooper ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Membrane Trafficking ◽  
Tyrosine Kinases ◽  
Sh3 Domain ◽  
Ph Domain ◽  
Gtpase Activating Protein ◽  
Binding Motifs ◽  
Hydrolysis Of ◽  
Adp Ribosylation Factor

ABSTRACT Membrane trafficking is regulated in part by small GTP-binding proteins of the ADP-ribosylation factor (Arf) family. Arf function depends on the controlled exchange and hydrolysis of GTP. We have purified and cloned two variants of a 130-kDa phosphatidylinositol 4,5-biphosphate (PIP2)-dependent Arf1 GTPase-activating protein (GAP), which we call ASAP1a and ASAP1b. Both contain a pleckstrin homology (PH) domain, a zinc finger similar to that found in another Arf GAP, three ankyrin (ANK) repeats, a proline-rich region with alternative splicing and SH3 binding motifs, eight repeats of the sequence E/DLPPKP, and an SH3 domain. Together, the PH, zinc finger, and ANK repeat regions possess PIP2-dependent GAP activity on Arf1 and Arf5, less activity on Arf6, and no detectable activity on Arl2 in vitro. The cDNA for ASAP1 was independently identified in a screen for proteins that interact with the SH3 domain of the tyrosine kinase Src. ASAP1 associates in vitro with the SH3 domains of Src family members and with the Crk adapter protein. ASAP1 coprecipitates with Src from cell lysates and is phosphorylated on tyrosine residues in cells expressing activated Src. Both coimmunoprecipitation and tyrosine phosphorylation depend on the same proline-rich class II Src SH3 binding site required for in vitro association. By directly interacting with both Arfs and tyrosine kinases involved in regulating cell growth and cytoskeletal organization, ASAP1 could coordinate membrane remodeling events with these processes.

scholarly journals Rna1p, a Ran/TC4 GTPase activating protein, is required for nuclear import.

1995 ◽  
Vol 130 (5) ◽  
pp. 1017-1026 ◽  
Author(s):  
A H Corbett ◽  
D M Koepp ◽  
G Schlenstedt ◽  
M S Lee ◽  
A K Hopper ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Gtpase Activating Protein ◽  
Direct Role ◽  
Green Fluorescent ◽  
Mutant Cells ◽  
Dose Dependent

The Saccharomyces cerevisiae gene, RNA1, encodes a protein with extensive homology to the mammalian Ran/TC4 GTPase activating protein. Using indirect immunofluorescence microscopy, we have demonstrated that rna1-1 mutant cells are defective in nuclear import of several proteins. The same result is obtained when nuclear import is examined in living cells using a nuclear protein fused to the naturally green fluorescent protein. These findings suggest a role for the Rna1p in trafficking of proteins across the nuclear membrane. To investigate this role more directly, an in vitro import assay that monitors the import of a fluorescently labeled substrate into the nuclei of semi-intact yeast cells was used. Import to the nucleus requires the addition of exogenous cytosol. Results indicate that, in contrast to wild-type cytosols, extracts made from rna1-1 mutant cells are unable to support import of the fluorescently labeled substrate into competent nuclei. Immunoblotting demonstrates that these mutant-derived extracts are depleted of Rna1p. However, when purified Rna1p is added back to these extracts the import activity is restored in a dose-dependent manner. These results demonstrate that Rna1p plays a direct role in the import of proteins into the nucleus.

scholarly journals Transcriptional perturbation of protein arginine methyltransferase-5 exhibits MTAP-selective oncosuppression

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sara Busacca ◽  
Qi Zhang ◽  
Annabel Sharkey ◽  
Alan G. Dawson ◽  
David A. Moore ◽  
...  
Keyword(s):  
Small Molecule ◽  
Selective Vulnerability ◽  
Dependent Manner ◽  
Histone H4 ◽  
Wild Type ◽  
Protein Arginine Methyltransferase ◽  
Arginine Methyltransferase ◽  
Methylthioadenosine Phosphorylase ◽  
Protein Arginine Methyltransferase 5

AbstractWe hypothesized that small molecule transcriptional perturbation could be harnessed to target a cellular dependency involving protein arginine methyltransferase 5 (PRMT5) in the context of methylthioadenosine phosphorylase (MTAP) deletion, seen frequently in malignant pleural mesothelioma (MPM). Here we show, that MTAP deletion is negatively prognostic in MPM. In vitro, the off-patent antibiotic Quinacrine efficiently suppressed PRMT5 transcription, causing chromatin remodelling with reduced global histone H4 symmetrical demethylation. Quinacrine phenocopied PRMT5 RNA interference and small molecule PRMT5 inhibition, reducing clonogenicity in an MTAP-dependent manner. This activity required a functional PRMT5 methyltransferase as MTAP negative cells were rescued by exogenous wild type PRMT5, but not a PRMT5E444Q methyltransferase-dead mutant. We identified c-jun as an essential PRMT5 transcription factor and a probable target for Quinacrine. Our results therefore suggest that small molecule-based transcriptional perturbation of PRMT5 can leverage a mutation-selective vulnerability, that is therapeutically tractable, and has relevance to 9p21 deleted cancers including MPM.

scholarly journals Estrogen Up-Regulates Hepatic Expression of Suppressors of Cytokine Signaling-2 and -3 in Vivo and in Vitro

Endocrinology ◽  
2004 ◽  
Vol 145 (12) ◽  
pp. 5525-5531 ◽  
Author(s):  
Gary M. Leong ◽  
Sofia Moverare ◽  
Jesena Brce ◽  
Nathan Doyle ◽  
Klara Sjögren ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Hepatoma Cells ◽  
Cytokine Signaling ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Wild Type ◽  
Hepatic Expression ◽  
Suppressors Of Cytokine Signaling

Abstract Suppressors of cytokine signaling (SOCS) are important negative regulators of cytokine action. We recently reported that estrogen stimulates SOCS-2 expression and inhibits GH signaling in kidney cells. The effects of estrogen on SOCS expression in other tissues are unclear. The aim of this study was to investigate in vivo and in vitro whether estrogen affected SOCS expression in the liver, a major target organ of GH. The in vivo hepatic effects of estrogen on ovariectomized mice lacking estrogen receptor (ER)-α, ERβ, or both and their wild-type littermates were examined by DNA microarray analysis. In vitro, the effects of estrogen on SOCS expression in human hepatoma cells were examined by reverse transcription quantitative PCR. Long-term (3 wk) estrogen treatment induced a 2- to 3-fold increase in hepatic expression of SOCS-2 and -3 in wild-type and ERβ knockout mice but not in those lacking ERα or both ER subtypes. Short-term treatment (at 24 h) increased the mRNA level of SOCS-3 but not SOCS-2. In cultured hepatoma cells, estrogen increased SOCS-2 and -3 mRNA levels by 2-fold in a time- and dose-dependent manner (P &lt; 0.05). Estrogen induced murine SOCS-3 promoter activity by 2-fold (P &lt; 0.05) in constructs containing a region between nucleotides −1862 and −855. Moreover, estrogen and GH had additive effects on the SOCS-3 promoter activity. In summary, estrogen, via ERα, up-regulated hepatic expression of SOCS-2 and -3, probably through transcriptional activation. This indicates a novel mechanism of estrogen regulation of cytokine action.

scholarly journals Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

2021 ◽  
Author(s):  
Fatima Amanat ◽  
Shirin Strohmeier ◽  
Wen-Hsin Lee ◽  
Sandhya Bangaru ◽  
Andrew B Ward ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Mouse Model ◽  
Neutralizing Antibodies ◽  
Dependent Manner ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

Abstract 5566: Recovery of Erk Signaling Restores Defective Angiogenesis and Arteriogenesis in Synectin-Deficient Animals

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Bin Ren ◽  
Arpita Mukhopadhyay* ◽  
Anthony A Lanahan ◽  
Zhen W Zhuang ◽  
Karen L Moodie ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Branching Morphogenesis ◽  
Erk Signaling ◽  
Dependent Manner ◽  
Wild Type ◽  
Erk Activation ◽  
Arterial Development ◽  
Constitutively Active

Background : Arterial morphogenesis is an important and poorly understood process. We have previously demonstrated that disruption of synectin gene expression in mice and zebrafish results in impaired arterial development and branching morphogenesis. Synectin null endothelial cells demonstrate reduced VEGF responsiveness in terms of migration, proliferation and differentiation and ERK-1/2 activation (Chittenden et al, Dev Cell 2006). Since ERK has been established as major participants in the regulation of cell growth and differentiation and Erk activation has been previously linked to arterial morphogenesis, we evaluated whether activation of Erk signaling in synectin disrupted mice and zebrafish as well as synectin KO arterial endothelial cells (ECs) would restore defective migration, arterial differentiation, angiogenesis and arteriogenesis. To stimulate ERK signaling we used partial inhibition of PI3-K activity to reduce Akt-dependent suppression of Raf1 activation or introduction of constitutively active ERK construct. Methods : In vitro studies were conducted with primary arterial ECs isolated from synectin wild type (WT) and knock out (KO) mice. In vivo studies were carried out in WT and synectin deficient mice and synectin knockdown zebrafish embryos. Results: Exposure of synectin −/− arterial EC to two selective PI3K inhibitors GS4898 or LY294002 in vitro restored ERK activation in a dose-dependent manner and returned cell migration and in vitro branching morphogenesis to wild type levels. Transduction of a constitutively active ERK construct in vitro or in a Matrigel model in vivo had similar effect. Systemic treatment of synectin −/− mice with GS4898 fully restored impaired angiogenesis and arterial morphogenesis in adult animals in the setting of hindlimb ischemia. Similar treatment nearly completely restored arterial development defects in zebrafish treated with a synectin morpholino. Conclusions: ERK activation plays a key role in arteriogenesis both in adult tissues and during embryonic development. Activation of compromised ERK-1/2 signaling may be a novel therapeutic intervention to stimulate arteriogenesis.

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells

1985 ◽  
Vol 5 (4) ◽  
pp. 902-905
Author(s):  
M Narkhammar ◽  
R Hand
Keyword(s):  
Cell Line ◽  
Nuclear Extract ◽  
Cell Extract ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Whole Cell ◽  
Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

scholarly journals The Pseudomonas aeruginosa product pyochelin interferes with Trypanosoma cruzi infection and multiplication in vitro

2020 ◽  
Vol 114 (7) ◽  
pp. 492-498 ◽  
Author(s):  
Gabriele Sass ◽  
Laura C Miller Conrad ◽  
Terrence-Thang H Nguyen ◽  
David A Stevens
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Iron Acquisition ◽  
Vero Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Current Standard ◽  
Cruzi Infection

Abstract Background Bacteria are sources of numerous molecules used in treatment of infectious diseases. We investigated effects of molecules produced by 26 Pseudomonas aeruginosa strains against infection of mammalian cell cultures with Trypanosoma cruzi, the aetiological agent of Chagas disease. Methods Vero cells were infected with T. cruzi in the presence of wild-type P. aeruginosa supernatants or supernatants of mutants with defects in the production of various virulence, quorum sensing and iron acquisition factors. Quantification of T. cruzi infection (percentage of infected cells) and multiplication (number of amastigotes per infected cell) was performed and cell viability was determined. Results Wild-type P. aeruginosa products negatively affected T. cruzi infection and multiplication in a dose-dependent manner, without evident toxicity for mammalian cells. PvdD/pchE mutation (loss of the P. aeruginosa siderophores pyoverdine and pyochelin) had the greatest impact on anti–T. cruzi activity. Negative effects on T. cruzi infection by pure pyochelin, but not pyoverdine, or other P. aeruginosa exoproducts studied, were quantitatively similar to the effects of benznidazole, the current standard therapy against T. cruzi. Conclusions The P. aeruginosa product pyochelin showed promising activity against T. cruzi and might become a new lead molecule for therapy development.

scholarly journals Loss of the F-Actin Binding and Vesicle-Associated Protein Comitin Leads to a Phagocytosis Defect

2002 ◽  
Vol 1 (6) ◽  
pp. 906-914 ◽  
Author(s):  
Thomas Schreiner ◽  
Martina R. Mohrs ◽  
Rosemarie Blau-Wasser ◽  
Alfred von Krempelhuber ◽  
Michael Steinert ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Gene Replacement ◽  
Actin Binding ◽  
Wild Type ◽  
Hyperosmotic Shock ◽  
Latex Beads ◽  
Knockout Mutants ◽  
Mutant Cells

ABSTRACT Comitin is an F-actin binding and membrane-associated protein from Dictyostelium discoideum, which is present on Golgi and vesicle membranes and changes its localization in response to agents affecting the cytoskeleton. To investigate its in vivo functions we have generated knockout mutants by gene replacement. Based on comitin's in vitro functions we examined properties related to vesicular transport and microfilament function. Whereas cell growth, pinocytosis, secretion, chemotaxis, motility, and development were unaltered, comitin-lacking cells were impaired in the early steps of phagocytosis of Saccharomyces cerevisiae particles and of Escherichia coli, whereas uptake of latex beads was unaffected. Furthermore, the lack of comitin positively affected survival of pathogenic bacteria. Mutant cells also showed an altered response to hyperosmotic shock in comparison to the wild type. The redistribution of comitin during hyperosmotic shock in wild-type cells and its presence on early phagosomes suggest a direct involvement of comitin in these processes.

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