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1.  Topological and Functional Properties of the Small GTPases Protein Interaction Network 
PLoS ONE  2012;7(9):e44882.
Small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran) regulate key cellular processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. A great deal of experimental evidence supports the existence of signaling cascades and feedback loops within and among the small GTPase subfamilies suggesting that these proteins function in a coordinated and cooperative manner. The interplay occurs largely through association with bi-partite regulatory and effector proteins but can also occur through the active form of the small GTPases themselves. In order to understand the connectivity of the small GTPases signaling routes, a systems-level approach that analyzes data describing direct and indirect interactions was used to construct the small GTPases protein interaction network. The data were curated from the Search Tool for the Retrieval of Interacting Genes (STRING) database and include only experimentally validated interactions. The network method enables the conceptualization of the overall structure as well as the underlying organization of the protein-protein interactions. The interaction network described here is comprised of 778 nodes and 1943 edges and has a scale-free topology. Rac1, Cdc42, RhoA, and HRas are identified as the hubs. Ten sub-network motifs are also identified in this study with themes in apoptosis, cell growth/proliferation, vesicle traffic, cell adhesion/junction dynamics, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase response, transcription regulation, receptor-mediated endocytosis, gene silencing, and growth factor signaling. Bottleneck proteins that bridge signaling paths and proteins that overlap in multiple small GTPase networks are described along with the functional annotation of all proteins in the network.
doi:10.1371/journal.pone.0044882
PMCID: PMC3441499  PMID: 23028658
2.  Phosphatidic acid signaling regulation of Ras superfamily of small guanosine triphosphatases 
Biochimica et biophysica acta  2009;1791(9):850-855.
Phosphatidic acid (PA) has been increasingly recognized as an important signaling lipid regulating cell growth and proliferation, membrane trafficking, and cytoskeletal reorganization. Recent studies indicate that the signaling PA generated from phospholipase D (PLD) and diacylglycerol kinase (DGK) plays critical roles in regulating the activity of some members of Ras superfamily of small guanosine triphosphatases (GTPases), such as Ras, Rac and Arf. Change of PA levels regulates the activity of small GTPases by modulating membrane localization and activity of small GTPase regulatory proteins, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). In addition, PA also targets some small GTPases to membranes by direct binding. This review summarizes the roles of PLD and DGK in regulating the activity of several Ras superfamily members and cellular processes they control. Some future directions and the implication of PA regulation of Ras small GTPases in pathology are also discussed.
doi:10.1016/j.bbalip.2009.05.013
PMCID: PMC2739575  PMID: 19540930
Phospholipids; Phosphatidic acid; Phospholipase D; Diacylglycerol kinase; Ras; Small GTPases
3.  Crosstalk of small GTPases at the Golgi apparatus 
Small GTPases  2012;3(2):80-90.
Small GTPases regulate a wide range of homeostatic processes such as cytoskeletal dynamics, organelle homeostasis, cell migration and vesicle trafficking, as well as in pathologic conditions such as carcinogenesis and metastatic spreading. Therefore, it is important to understand the regulation of small GTPase signaling, but this is complicated by the fact that crosstalk exists between different GTPase families and that we have to understand how they signal in time and space. The Golgi apparatus represents a hub for several signaling molecules and its importance in this field is constantly increasing. In this review we will discuss small GTPases signaling at the Golgi apparatus. Then, we will highlight recent work that contributed to a better understanding of crosstalk between different small GTPase families, with a special emphasis on their crosstalk at the Golgi apparatus. Finally, we will give a brief overview of available methods and tools to investigate spatio-temporal small GTPase crosstalk.
doi:10.4161/sgtp.19842
PMCID: PMC3408981  PMID: 22790194
Arf; Cdc42; Golgi apparatus; Rac; Ras; small GTPases; spatio-temporal signaling
4.  G-Protein-Coupled Receptor Interaction with Small GTPases 
Methods in enzymology  2013;522:97-108.
In addition to heterotrimeric G-proteins, Ras-like small GTPases are also involved in regulating physiological functions of the G-protein-coupled receptor (GPCR) superfamily. In particular, Rab and ARF GTPases function either as “traffic cops” to coordinate receptor targeting to specific locations or as “signal transducers” to directly mediate receptor signal propagation. As revealed in protein–protein interaction assays, GPCRs may use specific motifs to physically interact with small GTPases, providing important insights into the underlying molecular mechanisms. In this chapter, we describe coimmunoprecipitation and GST fusion protein pull-down approaches to study the GPCR-small GTPase interaction, by focusing on the interaction of α2B- and β2-adrenegic receptors with the small GTPases Rab8 and ARF1.
doi:10.1016/B978-0-12-407865-9.00006-6
PMCID: PMC3608118  PMID: 23374182
5.  Developmental expression of three small GTPases in the mouse eye 
Molecular Vision  2007;13:1144-1153.
Purpose
The small GTPases function as "molecular switches" by binding and releasing GTP to mediate downstream signaling effects. The Rho-family of GTPases is central in modulating cell differentiation and cytoskeletal changes. Since eye development requires comprehensive morphogenetic movements and extensive cellular differentiation, we hypothesize that different small GTPases may play important roles during morphogenesis of eye development. To explore this possibility, we examined the expression patterns of three major Rho-GTPases: RhoA, Rac1, and Cdc42 in embryonic, postnatal (one day after birth), and adult (two-month old) mouse eye.
Methods
Various ocular tissues were collected from embryonic, postnatal, and adult C57BL/6 mice. Western blots were conducted using total proteins extracted from cornea, retina, lens epithelial cells, and lens fiber cells of the adult mice or different fractions of rat lenses. Immunohistochemistry (IHC) was performed with 6 μm thick sections cut through the eye ball region of 11.5 pc, 14.5 pc, 17.5 pc, postnatal, and adult mice. Parallel controls were run using the rabbit preimmune and GTPase-specific antibodies blocked with saturating levels of corresponding peptide antigen.
Results
In the embryonic mouse eye, RhoA and Cdc42 expressions were initially detectable in all three compartments at 11.5 pc. However, Rac1 became easily detectable in these compartments at 14.5 pc. Increased levels of RhoA, Rac1, and Cdc42 were detected in the three compartments at 17.5 pc and the strongest signals for RhoA, Rac1, and Cdc42 were observed in the primary lens fiber cells at 17.5 pc. In the postnatal mouse eye, the three small GTPases were significantly expressed in both endothelial and epithelial cells of mouse cornea, epithelial cells of the ocular lens, photoreceptors, horizontal/amacrine/Muller's cells, and some ganglian cells of the retina. Much lower level of expression was observed in the corneal stroma fibroblasts, lens fiber cells, and the inner and outer plexiform layers of the mouse retina. In the adult mouse eye, all three Rho-GTPases were expressed in corneal epithelial cells and retina. However, only RhoA protein was detected in corneal endothelial cells and Rac1 protein detected in the ocular lens.
Conclusions
The strong expression of the three small GTPases in the cornea, lens, and retina of mouse eye at embryonic 17.5 pc and postnatal stage suggests their important functions for the morphogenesis of the different compartments of the mouse eye. Particularly, high levels of expression of RhoA, Rac1, and Cdc42 in embryonic lens fiber cells suggest their involvement in differentiation of primary lens fiber cells. In the adult mouse eye, all three Rho-GTPases seem to be involved in differentiation of corneal epithelial cells and retina, however, RhoA alone may be required for endothelial cell differentiation and Rac1 likely plays an important role in supporting continuous lens growth and maintenance of lens transparency.
PMCID: PMC2779149  PMID: 17653061
6.  Mutational analysis of the ribosome assembly GTPase RbgA provides insight into ribosome interaction and ribosome-stimulated GTPase activation 
Nucleic Acids Research  2013;41(5):3217-3227.
Ribosome biogenesis GTPase A protein (RbgA) is an essential GTPase required for the biogenesis of the 50S subunit in Bacillus subtilis. Homologs of RbgA are widely distributed in bacteria and eukaryotes and are implicated in ribosome assembly in the mitochondria, chloroplast and cytoplasm. Cells depleted of RbgA accumulate an immature large subunit that is missing key ribosomal proteins. RbgA, unlike many members of the Ras superfamily of GTPases, lacks a defined catalytic residue for carrying out guanosine triphosphate (GTP) hydrolysis. To probe RbgA function in ribosome assembly, we used a combined bioinformatics, genetic and biochemical approach. We identified a RNA-binding domain within the C-terminus of RbgA that is structurally similar to AmiR–NasR Transcription Anti-termination Regulator (ANTAR) domains, which are known to bind structured RNA. Mutation of key residues in the ANTAR domain altered RbgA association with the ribosome. We identified a putative catalytic residue within a highly conserved region of RbgA, His9, which is contained within a similar PGH motif found in elongation factor Tu (EF-Tu) that is required for GTP hydrolysis on interaction with the ribosome. Finally, our results support a model in which the GTPase activity of RbgA directly participates in the maturation of the large subunit rather than solely promoting dissociation of RbgA from the 50S subunit.
doi:10.1093/nar/gks1475
PMCID: PMC3597669  PMID: 23325847
7.  Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy? 
Nature reviews. Cancer  2010;10(12):842-857.
There is now considerable and increasing evidence for a causal role of aberrant activity of the Ras superfamily of small GTPases in human cancers. These GTPases act as GDP-GTP-regulated binary switches that control many fundamental cellular processes. A common mechanism of GTPase deregulation in cancer is the deregulated expression and/or activity of their regulatory proteins, guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound state and GTPase activating proteins (GAPs) that return the GTPase to its GDP-bound inactive state. We assess the association of GEFs and GAPs with cancer and their druggability for cancer therapeutics.
doi:10.1038/nrc2960
PMCID: PMC3124093  PMID: 21102635
8.  The Guanylate-Binding Proteins: Emerging Insights into the Biochemical Properties and Functions of This Family of Large Interferon-Induced Guanosine Triphosphatase 
Originally identified by their unusual ability to bind guanosine monophosphate (GMP) nucleotide agarose, the guanylate-binding proteins (GBPs) were used extensively to promote our understanding of interferon-induced gene transcription and as markers of interferon responsiveness. Structural and biochemical analyses of human GBP-1 subsequently demonstrated that the GBPs are a unique subfamily of guanosine triphosphatase (GTPases) that hydrolyze guanosine triphosphate (GTP) to both guanosine diphosphate (GDP) and GMP. As members of the larger dynamin superfamily of GTPases, GBPs exhibit such properties as nucleotide-dependent oligomerization and concentration-dependent GTPase activity. Recently, progress has been made in assigning functions to members of the GBP family. While many of these functions involve protection against intracellular pathogens, a growing number of them are not directly related to pathogen protection. It is currently unclear how the unusual properties of GBPs contribute to this growing list of functions. As future studies uncover the molecular mechanism(s) of action of the GBPs, we will gain a greater understanding of how individual GBPs can mediate what currently appears to be a divergent set of functions.
doi:10.1089/jir.2010.0102
PMCID: PMC3021356  PMID: 21142871
9.  The Rab GTPase family 
Genome Biology  2001;2(5):reviews3007.1-reviews3007.7.
The Rab family is part of the Ras superfamily of small GTPases. There are at least 60 Rab genes in the human genome, and a number of Rab GTPases are conserved from yeast to humans. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane traffic pathways. In the GTP-bound form, the Rab GTPases recruit specific sets of effector proteins onto membranes. Through their effectors, Rab GTPases regulate vesicle formation, actin- and tubulin-dependent vesicle movement, and membrane fusion.
PMCID: PMC138937  PMID: 11387043
10.  Small GTPases and cilia 
Protein & cell  2011;2(1):10.1007/s13238-011-1004-7.
Small GTPases are key molecular switches that bind and hydrolyze GTP in diverse membrane- and cytoskeleton-related cellular processes. Recently, mounting evidences have highlighted the role of various small GTPases, including the members in Arf/Arl, Rab, and Ran subfamilies, in cilia formation and function. Once overlooked as an evolutionary vestige, the primary cilium has attracted more and more attention in last decade because of its role in sensing various extracellular signals and the association between cilia dysfunction and a wide spectrum of human diseases, now called ciliopathies. Here we review recent advances about the function of small GTPases in the context of cilia, and the correlation between the functional impairment of small GTPases and ciliopathies. Understanding of these cellular processes is of fundamental importance for broadening our view of cilia development and function in normal and pathological states and for providing valuable insights into the role of various small GTPases in disease processes, and their potential as therapeutic targets.
doi:10.1007/s13238-011-1004-7
PMCID: PMC3858892  PMID: 21337006
Small GTPase; cilia; ciliopathy
11.  Ras Family of Small GTPases In Immunity And Inflammation 
Current opinion in pharmacology  2012;12(4):458-463.
The Ras superfamily of small GTPases is a group of more than 150 small G proteins, all of which share some degree of homology to the founding member Ras. These small GTPases function as molecular switches within cells, impacting nearly all cellular processes. The Ras superfamily can be further divided into several smaller subfamilies, with those proteins that most closely resemble Ras belonging to the Ras subfamily. While heavily studied within the field of cancer biology, the Ras family of proteins also plays cardinal roles in immunity and inflammation. Here we review the roles of these molecular switches in regulating immune cell homeostasis and functions.
doi:10.1016/j.coph.2012.02.003
PMCID: PMC3374043  PMID: 22401931
12.  Rap1 GTPases: An Emerging Role in the Cardiovasculature 
Life sciences  2011;88(15-16):645-652.
The Ras related GTPase Rap has been implicated in multiple cellular functions. A vital role for Rap GTPase in the cardiovasculature is emerging from recent studies. These small monomeric G proteins act as molecular switches, coupling extracellular stimulation to intracellular signaling through second messengers. This member of the Ras superfamily was once described as the transformation suppressor with the ability to ameliorate the Ras transformed phenotype; however, further studies uncovered a unique set of guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and effector proteins for Rap suggesting a more sophisticated role for this small GTPase. At least three different second messengers can activate Rap, namely cyclic AMP (cAMP), calcium and diacylglycerol. More recently, investigation of Rap in the cardiovasculature has revealed multiple pathways of regulation involving Rap in this system. Two closely related isoforms of Rap1 exist, 1A and 1B. Murine genetic models exist for both and have been described. Although thought at first to be functionally redundant, these isoforms have differing roles in the cardiovasculature. Activation of Rap1a and 1b in various cell types of the cardiovasculature lead to alterations in cell attachment, migration and cell junction formation. This review will focus on the role of these Rap1 GTPases in hematopoietic, endothelial, smooth muscle, and cardiac myocyte function, and conclude with their potential role in human disease.
doi:10.1016/j.lfs.2011.01.023
PMCID: PMC3090149  PMID: 21295042
Rap1; small GTPase; cAMP; Epac
13.  Advantages and limitations of cell-based assays for GTPase activation and regulation 
Cellular Logistics  2012;2(3):147-150.
Small GTPases of the Ras superfamily are important regulators of many cellular functions, including signal transduction, cytoskeleton assembly, metabolic regulation, organelle biogenesis and intracellular transport. Most GTPases act as binary switches, being “on” in the active, GTP-bound state and “off” in the inactive, GDP-bound state, and cycle between the two states with the aid of accessory proteins, referred to as guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). This review will focus on the ADP-ribosylation factors (Arfs), a family of G-proteins that are essential regulators of carrier vesicle formation during vesicular transport. As for most other GTPases, the Arfs themselves are vastly outnumbered by the proteins that regulate them, and a major focus in the field has been to define the functional relationships between individual GEFs and GAPs and their substrates at the cellular level. Over the years, a variety of methods have been developed to measure GTPase activation in vitro and in vivo. In vitro analysis will be discussed in the accompanying article by Randazzo and colleagues. Here we will focus on cell- and tissue-based assays and their advantages/disadvantages relative to cell-free systems.
doi:10.4161/cl.22045
PMCID: PMC3498073  PMID: 23181197
GTPase; Arf; GTP; GDP; GEF; GAP; effector; pulldown; FRET
14.  Inhibition of Rho and Rac Geranylgeranylation by Atorvastatin Is Critical for Preservation of Endothelial Junction Integrity 
PLoS ONE  2013;8(3):e59233.
Background
Small GTPases (guanosine triphosphate, GTP) are involved in many critical cellular processes, including inflammation, proliferation, and migration. GTP loading and isoprenylation are two important post-translational modifications of small GTPases, and are critical for their normal function. In this study, we investigated the role of post-translational modifications of small GTPases in regulating endothelial cell inflammatory responses and junctional integrity.
Methods and Results
Confluent human umbilical vein endothelial cell (HUVECs ) treated with atorvastatin demonstrated significantly decreased lipopolysaccharide (LPS)-mediated IL-6 and IL-8 generation. The inhibitory effect of atorvastatin (Atorva) was attenuated by co-treatment with 100 µM mevalonate (MVA) or 10 µM geranylgeranyl pyrophosphate (GGPP), but not by 10 µM farnesyl pyrophosphate (FPP). Atorvastatin treatment of HUVECs produced a time-dependent increase in GTP loading of all Rho GTPases, and induced the translocation of small Rho GTPases from the cellular membrane to the cytosol, which was reversed by 100 µM MVA and 10 µM GGPP, but not by 10 µM FPP. Atorvastatin significantly attenuated thrombin-induced HUVECs permeability, increased VE-cadherin targeting to cell junctions, and preserved junction integrity. These effects were partially reversed by GGPP but not by FPP, indicating that geranylgeranylation of small GTPases plays a major role in regulating endothelial junction integrity. Silencing of small GTPases showed that Rho and Rac, but not Cdc42, play central role in HUVECs junction integrity.
Conclusions
In conclusion, our studies show that post-translational modification of small GTPases plays a vital role in regulating endothelial inflammatory response and endothelial junction integrity. Atorvastatin increased GTP loading and inhibited isoprenylation of small GTPases, accompanied by reduced inflammatory response and preserved cellular junction integrity.
doi:10.1371/journal.pone.0059233
PMCID: PMC3596292  PMID: 23555637
15.  Signaling Through Rho GTPase Pathway as Viable Drug Target 
Current medicinal chemistry  2009;16(11):1355-1365.
Signaling through the Rho family of small GTPases has been increasingly investigated for their involvement in a wide variety of diseases such as cardiovascular, pulmonary, and neurological disorders as well as cancer. Rho GTPases are a subfamily of the Ras superfamily proteins which play essential roles in a number of biological processes, especially in the regulation of cell shape change, cytokinesis, cell adhesion, and cell migration. Many of these processes demonstrate a common theme: the rapid and dynamic reorganization of actin cytoskeleton of which Rho signaling has now emerged as a major switch control. The involvement of dynamic changes of Rho GTPases in disease states underscores the need to produce effective inhibitors for their therapeutic applications. Fasudil and Y-27632, with many newer additions, are two classes of widely used chemical compounds that inhibit Rho kinase (ROCK), an important downstream effector of RhoA subfamily GTPases. These inhibitors have been successful in many preclinical studies, indicating the potential benefit of clinical Rho pathway inhibition. On the other hand, except for Rac1 inhibitor NSC23766, there are few effective inhibitors directly targeting Rho GTPases, likely due to the lack of optimal structural information on individual Rho-RhoGEF, Rho-RhoGAP, or Rho-RhoGDI interaction to achieve specificity. Recently, LM11A-31 and other derivatives of peptide mimetic ligands for p75 neutrotrophoin receptor (p75NTR) show promising effects upstream of Rho GTPase signaling in neuronal regeneration. CCG-1423, a chemical compound showing profiles of inhibiting downstream of RhoA, is a further attempt for the development of novel pharmacological tools to disrupt Rho signaling pathway in cancer. Because of a rapidly growing number of studies deciphering the role of the Rho proteins in many diseases, specific and potent pharmaceutical modulators of various steps of Rho GTPase signaling pathway are critically needed to target for therapeutic intervention in cardiovascular disease, neurological disorders, and cancer progression.
PMCID: PMC3829470  PMID: 19355891
Rho GTPase; ROCK; Y-27632; fasudil; LM11A-31; NSC23766; CCG-1423
16.  A big new job for small GTPases 
Small GTPases  2012;3(3):159-162.
Members of the Ras superfamily of small guanosine triphosphatases (GTPases) function as key nodes within signaling networks in a remarkable range of cellular processes, including cell proliferation, differentiation, growth, cell-cell adhesion and apoptosis. We recently described a novel role for the Ras-like small GTPases Rap1 and Ral in regulating cortical polarity and spindle orientation during asymmetric neuroblast division in Drosophila. The participation of these proteins in promoting cell polarization seems to be a common theme throughout evolution.
doi:10.4161/sgtp.19631
PMCID: PMC3442801  PMID: 22647313
Asymmetric cell division; Ral; Rap1; cell polarity; small GTPases
17.  Nf1 RasGAP Inhibition of LIMK2 Mediates a New Cross-Talk between Ras and Rho Pathways 
PLoS ONE  2012;7(10):e47283.
Background
Ras GTPases mediate numerous biological processes through their ability to cycle between an inactive GDP-bound form and an active GTP-bound form. Guanine nucleotide exchange factors (GEFs) favor the formation of the active Ras-GTP, whereas GTPase activating proteins (GAPs) promote the formation of inactive Ras-GDP. Numerous studies have established complex signaling cross-talks between Ras GTPases and other members of the superfamily of small GTPases. GEFs were thought to play a major role in these cross-talks. However, recently GAPs were also shown to play crucial roles in these processes. Among RasGAPs, Nf1 is of special interest. Nf1 is responsible for the genetic disease Neurofibromatosis type I, and recent data strongly suggest that this RasGAP connects different signaling pathways.
Methodology/Principal Findings
In order to know if the RasGAP Nf1 might play a role in connecting Ras GTPases to other small GTPase pathways, we systematically looked for new partners of Nf1, by performing a yeast two-hybrid screening on its SecPH domain. LIMK2, a major kinase of the Rho/ROCK/LIMK2/cofilin pathway, was identified in this screening. We confirmed this interaction by co-immunoprecipitation experiments, and further characterized it. We also demonstrated its specificity: the close related homolog of LIMK2, LIMK1, does not interact with the SecPH domain of Nf1. We then showed that SecPH partially inhibits the kinase activity of LIMK2 on cofilin. Our results furthermore suggest a precise mechanism for this inhibition: in fact, SecPH would specifically prevent LIMK2 activation by ROCK, its upstream regulator.
Conclusions/Significance
Although previous data had already connected Nf1 to actin cytoskeleton dynamics, our study provides for the first time possible detailed molecular requirements of this involvement. Nf1/LIMK2 interaction and inhibition allows to directly connect neurofibromatosis type I to actin cytoskeleton remodeling, and provides evidence that the RasGAP Nf1 mediates a new cross-talk between Ras and Rho signaling pathways within the superfamily of small GTPases.
doi:10.1371/journal.pone.0047283
PMCID: PMC3474823  PMID: 23082153
18.  Signal recognition particle (SRP) and SRP receptor: A new paradigm for multi-state regulatory GTPases 
Biochemistry  2009;48(29):6696-6704.
The GTP-binding proteins or GTPases comprise a superfamily of proteins that provide molecular switches in numerous cellular processes. The ‘GTPase switch’ paradigm, in which a GTPase acts as a bimodal switch that is turned ‘on’ and ‘off’ by external regulatory factors, has been used to interpret the regulatory mechanism of many GTPases for over two decades. Nevertheless, recent work has unveiled an emerging class of ‘multi-state’ regulatory GTPases that do not adhere to this classical paradigm. Instead of relying on external nucleotide exchange factors or GTPase activating proteins to switch between the ‘on’ and ‘off’ states, these GTPases have the intrinsic ability to exchange nucleotides and to sense and respond to upstream and downstream factors. In contrast to the bimodal nature of the ‘GTPase switch’, these GTPases undergo multiple conformational rearrangements, allowing multiple regulatory points to be built into a complex biological process to ensure the efficiency and fidelity of the pathway. We suggest that these multi-state regulatory GTPases are uniquely suited to provide spatial and temporal control over complex cellular pathways that require multiple molecular events to occur in a highly coordinated fashion.
doi:10.1021/bi9006989
PMCID: PMC2883566  PMID: 19469550
19.  Identification and differential expression dynamics of peach small GTPases encoding genes during fruit development and ripening 
Journal of Experimental Botany  2010;61(10):2829-2842.
The function of monomeric GTPases of the RAS superfamily in fruit development and ripening has been partially characterized. Here the identification of peach (Prunus persica) small GTPases of the RAS superfamily expressed in fruit and the characterization of their expression profiles during fruit development are described. Extensive searches on expressed sequence tag (EST) databases led to the selection of a total of 24 genes from peach encoding proteins with significant similarity to Arabidopsis small GTPases. Sequence similarity analyses and identification of conserved motifs, diagnostic of specific RAS families and subfamilies, enabled bona fide assignment of fourteen PpRAB, seven PpARF/ARL/SAR, two PpROP and one PpRAN GTPases. Transcriptional expression profiles of peach monomeric GTPases, analysed by real-time quantitative reverse transcription-PCR, were obtained for mesocarp samples, collected in two consecutive years. Reproducible patterns of expression could be identified for five peach RAB-encoding genes (PpRABA1-1, PpRABA2, PpRABD2-1, PpRABD2-2, and PpRABC2), two ARFs (PpARFA1-1 and PpARLB1), and two ROPs (PpROP3 and PpROP4). Interestingly, the transient transcriptional up-regulation of PpARF genes and of PpRAB genes of the A and D clades, putatively controlling the exocytic delivery of cell wall components and modifying enzymes, appeared to coincide with peaks of growth speed and sugar accumulation and with the final phases of ripening. To our knowledge, this is the first description of the co-ordinated differential expression of a set of genes encoding small GTPases of the ARF and RAB families which takes place during key moments of fruit development and maturation.
doi:10.1093/jxb/erq116
PMCID: PMC2882273  PMID: 20501747
Fruit; GTPase; peach; ripening; trafficking; vesicle
20.  Optimization and stabilization of Rho small GTPase proteins for solution NMR studies 
Small GTPases  2011;2(6):295-304.
Rho GTPases of the Ras superfamily have important roles in regulating the organization of the actin filament system, morphogenesis and migration of cells. Structural details for these proteins are still emerging, and information on their dynamics in solution is much needed to understand the mechanisms underlying their signaling functions. This report reviews conditions for solution NMR studies of Rho GTPases and describes our optimization and stabilization of Rnd1 for such experiments. Rnd1 belongs to the Rnd protein subfamily branch of Rho small GTPases and functions in neurite outgrowth, dendrite development and in axon guidance. However, as we report here, solution NMR studies of this protein are challenging. Multiple methods have been employed to enhance the stability of Rnd1, including by cleavage of an N-terminal His expression tag and by addition of non-hydrolysable GMPPNP (β: γ-imidoguanosine 5'-triphosphate) nucleotide. Further stabilization of Rnd1 against aggregation was achieved through a structure informed point mutation while maintaining its conformation and binding affinity for a partner protein. The NMR spectrum of the optimized protein reveals significant improvement in NMR signal dispersion and intensity. This work paves the way for structural and protein-protein/protein-ligand interaction studies of Rnd1 by solution NMR and also provides a guide for optimization and stabilization of other Rho GTPases.
doi:10.4161/sgtp.19257
PMCID: PMC3337157  PMID: 22545226
expression; mutation; NMR; Rho GTPase; Rnd1; stabilization
21.  Cell motility 
The Ras proto-oncogenic proteins, prototypes of the small GTPases, work as molecular switches: they are active when bound to GTP and inactive when bound to GDP. A variety of evidence suggested that the Ras paradigm is not fully valid for the Rho-family of small GTPases. Indeed, permanent activation is not sufficient but it is rather the continuous oscillation between the GDP-bound and GTP-bound conformations (namely the GDP/GTP cycling or GTPase flux), that is required for Rho-GTPases to perform their biological functions and properly coordinate actin cytoskeleton reorganization. In our recent study, we show that Rac1 needs to cycle between the GDP and GTP states in order to efficiently control cell motility. Similarly, it was previously reported that GDP/GTP cycling is required by RhoA for cytokinesis and by Cdc42 for cell polarization. The future challenge is to understand why the GTPase flux is so important for the biological actions of Rho GTPases.
PMCID: PMC3306356  PMID: 22446552
22.  The Interplay between ROS and Ras GTPases: Physiological and Pathological Implications 
Journal of Signal Transduction  2011;2012:365769.
The members of the RasGTPase superfamily are involved in various signaling networks responsible for fundamental cellular processes. Their activity is determined by their guanine nucleotide-bound state. Recent evidence indicates that some of these proteins may be regulated by redox agents. Reactive oxygen species (ROSs) and reactive nitrogen species (RNSs) have been historically considered pathological agents which can react with and damage many biological macromolecules including DNA, proteins, and lipids. However, a growing number of reports have suggested that the intracellular production of ROS is tightly regulated and that these redox agents serve as signaling molecules being involved in a variety of cell signaling pathways. Numerous observations have suggested that some Ras GTPases appear to regulate ROS production and that oxidants function as effector molecules for the small GTPases, thus contributing to their overall biological function. Thus, redox agents may act both as upstream regulators and as downstream effectors of Ras GTPases. Here we discuss current understanding concerning mechanisms and physiopathological implications of the interplay between GTPases and redox agents.
doi:10.1155/2012/365769
PMCID: PMC3235814  PMID: 22175014
23.  Identification of a novel human Rho protein with unusual properties: GTPase deficiency and in vivo farnesylation. 
Molecular and Cellular Biology  1996;16(6):2689-2699.
We have identified a human Rho protein, RhoE, which has unusual structural and biochemical properties that suggest a novel mechanism of regulation. Within a region that is highly conserved among small GTPases, RhoE contains amino acid differences specifically at three positions that confer oncogenicity to Ras (12, 59, and 61). As predicted by these substitutions, which impair GTP hydrolysis in Ras, RhoE binds GTP but lacks intrinsic GTPase activity and is resistant to Rho-specific GTPase-activating proteins. Replacing all three positions in RhoE with conventional amino acids completely restores GTPase activity. In vivo, RhoE is found exclusively in the GTP-bound form, suggesting that unlike previously characterized small GTPases, RhoE may be normally maintained in an activated state. Thus, amino acid changes in Ras that are selected during tumorigenesis have evolved naturally in this Rho protein and have similar consequences for catalytic function. All previously described Rho family proteins are modified by geranylgeranylation, a lipid attachment required for proper membrane localization. In contrast, the carboxy-terminal sequence of RhoE predicts that, like Ras proteins, RhoE is normally farnesylated. Indeed, we have found that RhoE in farnesylated in vivo and that this modification is required for association with the plasma membrane and with an unidentified cellular structure that may play a role in adhesion. Thus, two unusual structural features of this novel Rho protein suggest a striking evolutionary divergence from the Rho family of GTPases.
PMCID: PMC231259  PMID: 8649376
24.  The taming of a Rab GTPase by Legionella pneumophila 
Small GTPases  2012;3(1):28-33.
Small GTPases of the Rab family represent an attractive target for microbial pathogens due to their role in controlling many aspects of intracellular cargo transport. Legionella pneumophila is an intravacuolar pathogen that survives inside host cells by manipulating protein trafficking pathways through a number of effector proteins secreted by the bacterium. These act as functional mimics of host proteins that modulate the activity of switch proteins such as guanosine triphosphatases (GTPases). L. pneumophila exploits the ER (endoplasmic reticulum)-to-Golgi vesicle transport pathway by modifying activity of Rab1, the GTPase regulating this pathway. This pathogen recruits Rab1 to the vacuole in which it resides, where effector proteins located on the surface of the vacuole regulate the activity status of Rab1 by mimicking the function of a guanine dissociation inhibitor (GDI) displacement factor, guanine exchange factor (GEF), or a GTPase-activating protein (GAP). In addition to these non-covalent modifications that alter the nucleotide binding state of Rab1, the bacterium also uses covalent modifications such as adenylylation (AMPylation) to control the dynamic of Rab1 on the Legionella-containing vacuole. Remarkably, AMPylation of Rab1 by SidM can be reversed by the L. pneumophila effector protein SidD, which exhibits de-AMPylation activity, demonstrating that L. pneumophila and related pathogens may utilize covalent modifications in order to transiently alter the activity of host proteins.
doi:10.4161/sgtp.18704
PMCID: PMC3398914  PMID: 22714414
AMPylation; de-AMPylation; GAP; GEF; Legionella; phosphocholination; Rab1
25.  Ubiquitination 
Small GTPases  2011;2(4):192-201.
The regulation of the small GTPases leading to their membrane localization has long been attributed to processing of their C-terminal CAAX box. As deregulation of many of these GTPases have been implicated in cancer and other disorders, prenylation and methylation of this CAAX box has been studied in depth as a possibility for drug targeting, but unfortunately, to date no drug has proved clinically beneficial. However, these GTPases also undergo other modifications that may be important for their regulation. Ubiquitination has long been demonstrated to regulate the fate of numerous cellular proteins and recently it has become apparent that many GTPases, along with their GAPs, GeFs and GDis, undergo ubiquitination leading to a variety of fates such as re-localization or degradation. in this review we focus on the recent literature demonstrating that the regulation of small GTPases by ubiquitination, either directly or indirectly, plays a considerable role in controlling their function and that targeting these modifications could be important for disease treatment.
doi:10.4161/sgtp.2.4.16707
PMCID: PMC3225908  PMID: 22145091
ubiquitination; Ras; Rho; USP; deubiquitination

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