scholarly journals Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay

10.3791/57646 ◽  
2018 ◽  
Author(s):  
Javad Alizadeh ◽  
Shahla Shojaei ◽  
Simone da Silva Rosa ◽  
Adel Rezaei Moghadam ◽  
Amir A. Zeki ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Small Gtpase ◽  
Membrane Fractionation ◽  
Gtp Binding

Small GTP-binding Protein RalA Associates with Weibel-Palade Bodies in Endothelial Cells

1999 ◽  
Vol 82 (09) ◽  
pp. 1177-1181 ◽  
Author(s):  
Hubert de Leeuw ◽  
Pauline Wijers-Koster ◽  
Jan van Mourik ◽  
Jan Voorberg
Keyword(s):  
Endothelial Cells ◽  
Binding Proteins ◽  
Binding Protein ◽  
Control Release ◽  
Small Gtpase ◽  
Gtp Binding Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Gtp Binding Proteins ◽  
Dense Granules ◽  
Gtp Binding

SummaryIn endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules, so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.

Small GTPase-regulated phospholipase D in granulocytes

1996 ◽  
Vol 74 (4) ◽  
pp. 459-467 ◽  
Author(s):  
Martin G. Houle ◽  
Sylvain Bourgoin
Keyword(s):  
Molecular Weight ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Tyrosine Kinases ◽  
Binding Proteins ◽  
Active Role ◽  
Small Gtpase ◽  
Low Molecular Weight ◽  
Gtp Binding Proteins ◽  
Gtp Binding

This review examines the functional role of phospholipase D in the neutrophil. Phospholipase D is emerging as an important component in the signal transduction pathways leading to granulocyte activation. Through the second messenger it produces, phosphatidic acid, phospholipase D plays an active role in the regulation of granulocyte NADPH oxidase activation and granular secretion. Many factors from both the cytosol and the membrane are necessary for maximal phospholipase D activation. This paper will focus on the regulation of phospholipase D by low molecular weight GTP-binding proteins, tyrosine kinases, and protein kinase C.Key words: phospholipase D, low molecular weight GTP-binding proteins, tyrosine kinases, protein kinase C, granulocytes.

The Small GTPase Superfamily in Plants: A Conserved Regulatory Module with Novel Functions

2020 ◽  
Vol 71 (1) ◽  
pp. 247-272
Author(s):  
Erik Nielsen
Keyword(s):  
Membrane Trafficking ◽  
Binding Proteins ◽  
Model Organism ◽  
Nucleocytoplasmic Transport ◽  
Small Gtpase ◽  
Gtp Binding Proteins ◽  
Cellular Processes ◽  
Gtp Binding ◽  
Regulatory Module ◽  
Transport Vesicle

Small GTP-binding proteins represent a highly conserved signaling module in eukaryotes that regulates diverse cellular processes such as signal transduction, cytoskeletal organization and cell polarity, cell proliferation and differentiation, intracellular membrane trafficking and transport vesicle formation, and nucleocytoplasmic transport. These proteins function as molecular switches that cycle between active and inactive states, and this cycle is linked to GTP binding and hydrolysis. In this review, the roles of the plant complement of small GTP-binding proteins in these cellular processes are described, as well as accessory proteins that control their activity, and current understanding of the functions of individual members of these families in plants—with a focus on the model organism Arabidopsis—is presented. Some potential novel roles of these GTPases in plants, relative to their established roles in yeast and/or animal systems, are also discussed.

Nitric oxide-induced inhibition of smooth muscle cell proliferation involves S-nitrosation and inactivation of RhoA

2007 ◽  
Vol 292 (2) ◽  
pp. C824-C831 ◽  
Author(s):  
Brian S. Zuckerbraun ◽  
Detcho A. Stoyanovsky ◽  
Rajib Sengupta ◽  
Richard A. Shapiro ◽  
Brett A. Ozanich ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Small Gtpase ◽  
Erk Signaling ◽  
Erk Pathway ◽  
Western Blots ◽  
Gtp Binding ◽  
Rhoa Protein

Nitric oxide (NO) acts as a vasoregulatory molecule that inhibits vascular smooth muscle cell (SMC) proliferation. Studies have illustrated that NO inhibits SMC proliferation via the extracellular signal-regulated kinase (ERK) pathway, leading to increased protein levels of the cyclin-dependent kinase inhibitor p21Waf1/Cip1. The ERK pathway can be pro- or antiproliferative, and it has been demonstrated that the activation status of the small GTPase RhoA determines the proliferative fate of ERK signaling, whereby inactivation of RhoA influences ERK signaling to increase p21Waf1/Cip1 and inhibit proliferation. The purpose of these investigations was to examine the effect of NO on RhoA activation/ S-nitrosation and to test the hypothesis that inhibition of SMC proliferation by NO is dependent on inactivation of RhoA. NO decreases activation of RhoA, as demonstrated by RhoA GTP-binding assays, affinity precipitation, and phalloidin staining of the actin cytoskeleton. Additionally, these effects are independent of cGMP. NO decreases SMC proliferation, and gene transfer of constitutively active RhoA (RhoA63L) diminished the antiproliferative effects of NO, as determined by thymidine incorporation. Western blots of p21Waf1/Cip1 correlated with changes in proliferation. S-nitrosation of recombinant RhoA protein and immunoprecipitated RhoA was demonstrated by Western blotting for nitrosocysteine and by measurement of NO release. Furthermore, NO decreases GTP loading of recombinant RhoA protein. These findings indicate that inactivation of RhoA plays a role in NO-mediated SMC antiproliferation and that S-nitrosation is associated with decreased GTP binding of RhoA. Nitrosation of RhoA and other proteins likely contributes to cGMP-independent effects of NO.

scholarly journals Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo

2002 ◽  
Vol 367 (3) ◽  
pp. 617-628 ◽  
Author(s):  
Maria L. HENRIKSSON ◽  
Charlotta SUNDIN ◽  
Anna L. JANSSON ◽  
Åke FORSBERG ◽  
Ruth H. PALMER ◽  
...  
Keyword(s):  
Small Gtpases ◽  
Small Gtpase ◽  
Eukaryotic Cells ◽  
Gtpase Activating Protein ◽  
Exoenzyme S ◽  
Adp Ribosylation ◽  
Intracellular Targeting ◽  
Gtp Binding ◽  
Adp Ribosyltransferase

Intracellular targeting of the Pseudomonas aeruginosa toxins exoenzyme S (ExoS) and exoenzyme T (ExoT) initially results in disruption of the actin microfilament structure of eukaryotic cells. ExoS and ExoT are bifunctional cytotoxins, with N-terminal GTPase-activating protein (GAP) and C-terminal ADP-ribosyltransferase activities. We show that ExoS can modify multiple GTPases of the Ras superfamily in vivo. In contrast, ExoT shows no ADP-ribosylation activity towards any of the GTPases tested in vivo. We further examined ExoS targets in vivo and observed that ExoS modulates the activity of several of these small GTP-binding proteins, such as Ras, Rap1, Rap2, Ral, Rac1, RhoA and Cdc42. We suggest that ExoS is the major ADP-ribosyltransferase protein modulating small GTPase function encoded by P. aeruginosa. Furthermore, we show that the GAP activity of ExoS abrogates the activation of RhoA, Cdc42 and Rap1.

scholarly journals Identification of a Small GTPase Inhibitor Using a High-Throughput Flow Cytometry Bead-Based Multiplex Assay

2009 ◽  
Vol 15 (1) ◽  
pp. 10-20 ◽  
Author(s):  
Zurab Surviladze ◽  
Anna Waller ◽  
Yang Wu ◽  
Elsa Romero ◽  
Bruce S. Edwards ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Small Molecule ◽  
High Throughput ◽  
False Negative ◽  
Small Gtpases ◽  
Multiplex Assay ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Gtp Binding

Small GTPases are key regulators of cellular activity and represent novel targets for the treatment of human diseases using small-molecule inhibitors. The authors describe a multiplex, flow cytometry bead-based assay for the identification and characterization of inhibitors or activators of small GTPases. Six different glutathione-S-transferase (GST)—tagged small GTPases were bound to glutathione beads, each labeled with a different red fluorescence intensity. Subsequently, beads bearing different GTPase were mixed and dispensed into 384-well plates with test compounds, and fluorescent—guanosine triphosphate (GTP) binding was used as the readout. This novel multiplex assay allowed the authors to screen a library of almost 200,000 compounds and identify more than 1200 positive compounds, which were further verified by dose-response analyses, using 6- to 8-plex assays. After the elimination of false-positive and false-negative compounds, several small-molecule families with opposing effects on GTP binding activity were identified. The authors detail the characterization of MLS000532223, a general inhibitor that prevents GTP binding to several GTPases in a dose-dependent manner and is active in biochemical and cell-based secondary assays. Live-cell imaging and confocal microscopy studies revealed the inhibitor-induced actin reorganization and cell morphology changes, characteristic of Rho GTPases inhibition. Thus, high-throughput screening via flow cytometry provides a strategy for identifying novel compounds that are active against small GTPases.

scholarly journals Small sequence variations between two mammalian paralogs of the small GTPase SAR1 underlie functional differences in coat protein complex II assembly

2020 ◽  
Vol 295 (25) ◽  
pp. 8401-8412 ◽  
Author(s):  
David B. Melville ◽  
Sean Studer ◽  
Randy Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Binding Site ◽  
Protein Complex ◽  
Small Gtpase ◽  
The Other ◽  
Complex Ii ◽  
Gtp Binding ◽  
Lipoprotein Secretion ◽  
Coat Protein Complex

Vesicles that are coated by coat protein complex II (COPII) are the primary mediators of vesicular traffic from the endoplasmic reticulum to the Golgi apparatus. Secretion-associated Ras-related GTPase 1 (SAR1) is a small GTPase that is part of COPII and, upon GTP binding, recruits the other COPII proteins to the endoplasmic reticulum membrane. Mammals have two SAR1 paralogs that genetic data suggest may have distinct physiological roles, e.g. in lipoprotein secretion in the case of SAR1B. Here we identified two amino acid clusters that have conserved SAR1 paralog–specific sequences. We observed that one cluster is adjacent to the SAR1 GTP-binding pocket and alters the kinetics of GTP exchange. The other cluster is adjacent to the binding site for two COPII components, SEC31 homolog A COPII coat complex component (SEC31) and SEC23. We found that the latter cluster confers to SAR1B a binding preference for SEC23A that is stronger than that of SAR1A for SEC23A. Unlike SAR1B, SAR1A was prone to oligomerize on a membrane surface. SAR1B knockdown caused loss of lipoprotein secretion, overexpression of SAR1B but not of SAR1A could restore secretion, and a divergent cluster adjacent to the SEC31/SEC23-binding site was critical for this SAR1B function. These results highlight that small primary sequence differences between the two mammalian SAR1 paralogs lead to pronounced biochemical differences that significantly affect COPII assembly and identify a specific function for SAR1B in lipoprotein secretion, providing insights into the mechanisms of large cargo secretion that may be relevant for COPII-related diseases.

scholarly journals ARL15 modulates magnesium homeostasis through N-glycosylation of CNNMs

2020 ◽  
Author(s):  
Yevgen Zolotarov ◽  
Chao Ma ◽  
Irene González-Recio ◽  
Serge Hardy ◽  
Gijs Franken ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Golgi Apparatus ◽  
In Silico ◽  
Small Gtpase ◽  
Negative Regulator ◽  
Magnesium Homeostasis ◽  
Gtp Binding ◽  
Carboxyl Terminal ◽  
Cbs Domains ◽  
Adp Ribosylation Factor

ABSTRACTCyclin M (CNNM1-4) proteins maintain cellular and body magnesium (Mg2+) homeostasis. Using various biochemical approaches, we have identified members of the CNNM family as direct interacting partners of ADP-ribosylation factor-like protein 15 (ARL15), a small GTP-binding protein. ARL15 interacts with CNNMs at their carboxyl-terminal conserved cystathionine-β-synthase (CBS) domains. In silico modeling of the interaction using the reported structures of both CNNM2 and ARL15 supports that the small GTPase specifically binds the CBS1 domain. Immunocytochemical experiments demonstrate that CNNM2 and ARL15 co-localize in the kidney, with both proteins showing subcellular localization in the Golgi-apparatus. Most importantly, we found that ARL15 is required for forming complex N-glycosylation of CNNMs. Overexpression of ARL15 promotes complex N-glycosylation of CNNM3. Mg2+ uptake experiments with a stable isotope demonstrate that there is a significant increase of 25Mg2+ uptake upon knockdown of ARL15 in multiple kidney cancer cell lines. Altogether, our results establish ARL15 as a novel negative regulator of Mg2+ transport by promoting the complex N-glycosylation of CNNMs.

scholarly journals SAR1 paralogs differ biochemically in assembly of the COPII coat

2020 ◽  
Author(s):  
David B. Melville ◽  
Sean Studer ◽  
Randy Schekman
Keyword(s):  
Binding Site ◽  
Membrane Surface ◽  
Binding Pocket ◽  
Small Gtpase ◽  
The Other ◽  
The Novel ◽  
Vesicular Traffic ◽  
Gtp Binding ◽  
Binding Preference ◽  
Lipoprotein Secretion

ABSTRACTCOPII-coated vesicles are the primary mediators of vesicular traffic from the ER to the Golgi apparatus. SAR1 is a small GTPase, which, upon GTP binding, recruits the other COPII proteins to the ER membrane. In mammals, there are two SAR1 paralogs which genetic data suggest may have distinct physiological roles, e.g. in lipoprotein secretion for SAR1B. We identified two clusters of amino acids that have conserved, paralog-specific sequences. One cluster is adjacent to the SAR1 GTP-binding pocket and alters the kinetics of GTP exchange. The other cluster is adjacent to the binding site of COPII components SEC31 and SEC23. We found that the latter cluster confers a SEC23A binding preference to SAR1B over SAR1A. In contrast to SAR1B, SAR1A is prone to oligomerize on a membrane surface. Importantly, in relation to its physiological function, SAR1B, but not SAR1A, can compensate for loss of SAR1B in lipoprotein secretion. The SEC31/SEC23-binding site-adjacent divergent cluster is critical for this function. These data identify the novel paralog-specific function for SAR1B, and provide insights into the mechanisms of large cargo secretion and COPII related diseases.

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