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Rapid degradation of dominant-negative Rab27 proteins in vivo precludes their use in transgenic mouse models
José S Ramalho, Ross Anders, Gesine B Jaissle, Mathias W Seeliger, Clare Huxley, Miguel C Seabra
BMC Cell Biology , 2002, DOI: 10.1186/1471-2121-3-26
Abstract: To investigate this hypothesis, we generated several lines of dominant-negative, constitutively-active and wild-type Rab27a (and Rab27b) transgenic mice whose expression was driven either by the pigment cell-specific tyrosinase promoter or the ubiquitous β-actin promoter. High levels of mRNA and protein were observed in transgenic lines expressing wild-type or constitutively active Rab27a and Rab27b. However, only modest levels of transgenic protein were expressed. Pulse-chase experiments suggest that the dominant-negative proteins, but not the constitutively-active or wild type proteins, are rapidly degraded. Consistently, no significant phenotype was observed in our transgenic lines. Coat-colour was normal, indicating normal Rab27a activity. Retinal function as determined by fundoscopy, angiography, electroretinography and histology was also normal.We suggest that the instability of the dominant-negative mutant Rab27 proteins in vivo precludes the use of this approach to generate mouse models of disease caused by Rab27 GTPases.Membrane traffic in eukaryotic cells is mediated by vesicular carriers, which bud from a donor compartment, are targeted to, and fuse with the appropriate acceptor membrane. The Rab family of Ras-like GTPases is known to play a crucial role controlling these mechanisms [1,2]. Among Rabs, the Rab27 subfamily consists of two isoforms, Rab27a (previously designated Ram) [3] and Rab27b (previously designated c25KG) [4].In order to function in membrane traffic, Rabs require the covalent addition of one or two C20 geranylgeranyl groups [5,6]. Geranylgeranylation serves as a membrane-anchoring device in Rab proteins [5]. This reaction is complex and occurs in several steps. Newly synthesised Rab proteins first associate with Rab Escort Protein (REP) and form a stable 1:1 complex [7,8]. The complex then serves as a substrate for Rab geranylgeranyl transferase to catalyse geranylgeranylation via thioether bonds to carboxy-terminal cysteine residues i
Insights into the classification of small GTPases
Dominik Heider, Sascha Hauke, Martin Pyka, et al
Advances and Applications in Bioinformatics and Chemistry , 2010, DOI: http://dx.doi.org/10.2147/AABC.S8891
Abstract: sights into the classification of small GTPases Original Research (6567) Total Article Views Authors: Dominik Heider, Sascha Hauke, Martin Pyka, et al Published Date May 2010 Volume 2010:3 Pages 15 - 24 DOI: http://dx.doi.org/10.2147/AABC.S8891 Dominik Heider1, Sascha Hauke3, Martin Pyka4, Daniel Kessler2 1Department of Bioinformatics, Center for Medical Biotechnology, 2Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Essen, Germany; 3Institute of Computer Science, University of Münster, Münster, Germany; 4Interdisciplinary Center for Clinical Research, University Hospital of Münster, Münster, Germany Abstract: In this study we used a Random Forest-based approach for an assignment of small guanosine triphosphate proteins (GTPases) to specific subgroups. Small GTPases represent an important functional group of proteins that serve as molecular switches in a wide range of fundamental cellular processes, including intracellular transport, movement and signaling events. These proteins have further gained a special emphasis in cancer research, because within the last decades a huge variety of small GTPases from different subgroups could be related to the development of all types of tumors. Using a random forest approach, we were able to identify the most important amino acid positions for the classification process within the small GTPases superfamily and its subgroups. These positions are in line with the results of earlier studies and have been shown to be the essential elements for the different functionalities of the GTPase families. Furthermore, we provide an accurate and reliable software tool (GTPasePred) to identify potential novel GTPases and demonstrate its application to genome sequences.
Insights into the classification of small GTPases  [cached]
Dominik Heider,Sascha Hauke,Martin Pyka,et al
Advances and Applications in Bioinformatics and Chemistry , 2010,
Abstract: Dominik Heider1, Sascha Hauke3, Martin Pyka4, Daniel Kessler21Department of Bioinformatics, Center for Medical Biotechnology, 2Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Essen, Germany; 3Institute of Computer Science, University of Münster, Münster, Germany; 4Interdisciplinary Center for Clinical Research, University Hospital of Münster, Münster, GermanyAbstract: In this study we used a Random Forest-based approach for an assignment of small guanosine triphosphate proteins (GTPases) to specific subgroups. Small GTPases represent an important functional group of proteins that serve as molecular switches in a wide range of fundamental cellular processes, including intracellular transport, movement and signaling events. These proteins have further gained a special emphasis in cancer research, because within the last decades a huge variety of small GTPases from different subgroups could be related to the development of all types of tumors. Using a random forest approach, we were able to identify the most important amino acid positions for the classification process within the small GTPases superfamily and its subgroups. These positions are in line with the results of earlier studies and have been shown to be the essential elements for the different functionalities of the GTPase families. Furthermore, we provide an accurate and reliable software tool (GTPasePred) to identify potential novel GTPases and demonstrate its application to genome sequences.Keywords: cancer, machine learning, classification, Random Forests, proteins
GTPases and the origin of the ribosome
Hyman Hartman, Temple F Smith
Biology Direct , 2010, DOI: 10.1186/1745-6150-5-36
Abstract: 1) The Elongation Factors of the Archaea based on structural considerations of the domains have the following evolutionary path: EF1→ aeIF2 → EF2. The evolution of the aeIF5b was a later event; 2) the Elongation Factors of the Bacteria based on the topological considerations of the GTPase domain have a similar evolutionary path: EFTu→ IF→2→EFG. These evolutionary sequences reflect the evolution of the LSU followed by the SSU to form the ribosome; 3) the OB-fold IF1 is a mimic of an ancient tRNA minihelix.The evolution of translational GTPases of both the Archaea and Bacteria point to the evolution of the ribosome. The elongation factors, EFTu/EF1, began as a Ras-like GTPase bringing the activated minihelix tRNA to the Large Subunit Unit. The initiation factors and elongation factor would then have evolved from the EFTu/EF1 as the small subunit was added to the evolving ribosome. The SRP has an SRP54 GTPase and a specific RNA fold in its RNA component similar to the PTC. We consider the SRP to be a remnant of an ancient form of an LSU bound to a membrane.This article was reviewed by George Fox, Leonid Mirny and Chris Sander.This study is on the origin and evolution of the ribosome Large Subunit (LSU) and Small Subunit (SSU) as reflected in the structure of the universal ribosomal translational GTPases. This continues an earlier study [1] on the origin of the ribosome as reflected in the structure of the ribosomal proteins. That study led to the conclusion that the LSU began as a Peptide Transfer Center (PTC)-like RNA fold on a peptide membrane. The subsequent evolution of the LSU was only later joined by the addition of an evolving SSU.Recent papers by Bokov and Steinberg [2], Hury et al. [3], and Fox and Naik [4] consider the evolution of the LSU of the ribosome to start from a PTC-like RNA. It is becoming clear that the evolution of the ribosome begins with the evolution of the LSU. This should be reflected in the evolution of the GTPases associated with the riboso
The Interplay between ROS and Ras GTPases: Physiological and Pathological Implications  [PDF]
Elisa Ferro,Luca Goitre,Saverio Francesco Retta,Lorenza Trabalzini
Journal of Signal Transduction , 2012, DOI: 10.1155/2012/365769
Abstract: 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. 1. Introduction The Ras GTPase superfamily includes low molecular weight GTP-binding and hydrolyzing (GTPases) proteins that act as molecular switches by coupling extracellular signals to different cellular responses, thus controlling cellular signaling pathways responsible for growth, migration, adhesion, cytoskeletal integrity, survival, and differentiation. The three human Ras proteins, H-Ras, N-Ras, and K-Ras, are the founding members of this large superfamily of small GTPases comprising over 150 human members with evolutionarily conserved orthologs found in Drosophila, C. elegans, S. cerevisiae, S. pombe, Dictyostelium, and plants. This superfamily is divided into families and subfamilies on the basis of sequence and functional similarities (Table 1). The five major families are Ras, Rho, Rab, Arf, and Ran [1]. In addition to the different Ras isoforms, the Ras family includes Rap, R-Ras, Ral, and Rheb proteins, also regulating signaling networks. Rho GTPase family includes the well-characterized family members Rac1, RhoA, and Cdc42, each of which is associated with unique phenotypes and functions [2–4]. Rab proteins comprise the largest branch of superfamily and regulate intracellular vesicular transport and trafficking of proteins. Like the Rab proteins, Arf family proteins are involved in regulation of vesicular transport. The Ran protein is the most abundant
Phylogenetic distribution of translational GTPases in bacteria
T?nu Margus, Maido Remm, Tanel Tenson
BMC Genomics , 2007, DOI: 10.1186/1471-2164-8-15
Abstract: To achieve accurate protein identification and grouping we have developed a method combining searches with Hidden Markov Model profiles and tree based grouping. We found all the genes for translational GTPases in 191 fully sequenced bacterial genomes. The protein sequences were grouped into nine subfamilies.Analysis of the results shows that three translational GTPases, the translation factors EF-Tu, EF-G and IF2, are present in all organisms examined. In addition, several copies of the genes encoding EF-Tu and EF-G are present in some genomes. In the case of multiple genes for EF-Tu, the gene copies are nearly identical; in the case of multiple EF-G genes, the gene copies have been considerably diverged. The fourth translational GTPase, LepA, the function of which is currently unknown, is also nearly universally conserved in bacteria, being absent from only one organism out of the 191 analyzed. The translation regulator, TypA, is also present in most of the organisms examined, being absent only from bacteria with small genomes.Surprisingly, some of the well studied translational GTPases are present only in a very small number of bacteria. The translation termination factor RF3 is absent from many groups of bacteria with both small and large genomes. The specialized translation factor for selenocysteine incorporation – SelB – was found in only 39 organisms. Similarly, the tetracycline resistance proteins (Tet) are present only in a small number of species.Proteins of the CysN/NodQ subfamily have acquired functions in sulfur metabolism and production of signaling molecules. The genes coding for CysN/NodQ proteins were found in 74 genomes. This protein subfamily is not confined to Proteobacteria, as suggested previously but present also in many other groups of bacteria.Four of the translational GTPase subfamilies (IF2, EF-Tu, EF-G and LepA) are represented by at least one member in each bacterium studied, with one exception in LepA. This defines the set of translation
Rho and Ras GTPases in semaphorin-mediated neuronal development  [PDF]
Lifei Fan, Morigen  
Advances in Bioscience and Biotechnology (ABB) , 2013, DOI: 10.4236/abb.2013.41A020
Abstract:

Neurons are highly polarized cells with a single long axon and multiple dendrites, all of which are actinrich structures. The precise regulation of neuronal cell morphology is a fundamental aspect of neurobiology. The major role of Rho GTPases, which is conserved in all eukaryotes, is to regulate the actin and microtubule cytoskeleton. Therefore theRhoGTPases are key regulators of neuronal morphology during development besides their canonical functions in actin cytoskeletal regulation, cell migration and cell cycle progression. Semaphorins are a family of secreted or transmembrane proteins, which function through their receptor plexins and/or neuropilins to act as the repulsive or attractive guidance cues for axons and dendrites. It has been demonstrated that the fully activetion of plexins appears to be dependent on the binding of RhoGTPases to theRhobinding domain (RBD) and Semaphorin to the extracellular region. Here, we summarize the functions of the small Rho GTPases in two well-studied vertebrate Semaphorins, Sema3Aand Sema4D; and the potential roles of the small Rho GTPases in some poorly-studied vertebrate Semaphorins Sema5A, Sema6Aand Sema7A. We also summarize the functions of different members of Ras family, R-Ras, M-Ras and Rap, in Semaphorin signalling pathways as well.

Separable states can be used to distribute entanglement  [PDF]
T. S. Cubitt,F. Verstraete,W. Dur,J. I. Cirac
Physics , 2003, DOI: 10.1103/PhysRevLett.91.037902
Abstract: We show that no entanglement is necessary to distribute entanglement; that is, two distant particles can be entangled by sending a third particle that is never entangled with the other two. Similarly, two particles can become entangled by continuous interaction with a highly mixed mediating particle that never itself becomes entangled. We also consider analogous properties of completely positive maps, in which the composition of two separable maps can create entanglement.
Novel Split-Luciferase-Based Genetically Encoded Biosensors for Noninvasive Visualization of Rho GTPases  [PDF]
Weibing Leng, Xiaohui Pang, Hongwei Xia, Mingxing Li, Liu Chen, Qiulin Tang, Dandan Yuan, Ronghui Li, Libo Li, Fabao Gao, Feng Bi
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0062230
Abstract: Rho family GTPases are critical regulators of many important cellular processes and the dysregulation of their activities is implicated in a variety of human diseases including oncogenesis and propagation of malignancy. The traditional methods, such as “pull-down” or two-hybrid procedures, are poorly suited to dynamically evaluate the activity of Rho GTPases, especially in living mammalian cells. To provide a novel alternative approach to analyzing Rho GTPase-associated signaling pathways in vivo, we developed a series of bioluminescent biosensors based on the genetically engineered firefly luciferase. These split-luciferase-based biosensors enable non-invasive visualization and quantification of the activity of Rho GTPases in living subjects. The strategy is to reasonably split the gene of firefly luciferase protein into two inactive fragments and then respectively fuse the two fragments to Rho GTPase and the GTPase-binding domain (GBD) of the specific effector. Upon Rho GTPase interacting with the binding domain in a GTP-dependent manner, these two luciferase fragments are brought into close proximity, leading to luciferase reconstitution and photon production in the presence of the substrate. Using these bimolecular luminescence complementation (BiLC) biosensors, we successfully visualized and quantified the activities of the three best characterized Rho GTPases by measuring the luminescence in living cells. We also experimentally investigated the sensitivity of these Rho GTPase biosensors to upstream regulatory proteins and extracellular ligands without lysing cells and doing labor-intensive works. By virtue of the unique functional characteristics of bioluminescence imaging, the BiLC-based biosensors provide an enormous potential for in vivo imaging of Rho GTPase signaling pathways and high-throughput screening of therapeutic drugs targeted to Rho GTPases and (or) upstream molecules in the near future.
Thousands of Rab GTPases for the Cell Biologist  [PDF]
Yoan Diekmann ,Elsa Seixas,Marc Gouw,Filipe Tavares-Cadete,Miguel C. Seabra,José B. Pereira-Leal
PLOS Computational Biology , 2011, DOI: 10.1371/journal.pcbi.1002217
Abstract: Rab proteins are small GTPases that act as essential regulators of vesicular trafficking. 44 subfamilies are known in humans, performing specific sets of functions at distinct subcellular localisations and tissues. Rab function is conserved even amongst distant orthologs. Hence, the annotation of Rabs yields functional predictions about the cell biology of trafficking. So far, annotating Rabs has been a laborious manual task not feasible for current and future genomic output of deep sequencing technologies. We developed, validated and benchmarked the Rabifier, an automated bioinformatic pipeline for the identification and classification of Rabs, which achieves up to 90% classification accuracy. We cataloged roughly 8.000 Rabs from 247 genomes covering the entire eukaryotic tree. The full Rab database and a web tool implementing the pipeline are publicly available at www.RabDB.org. For the first time, we describe and analyse the evolution of Rabs in a dataset covering the whole eukaryotic phylogeny. We found a highly dynamic family undergoing frequent taxon-specific expansions and losses. We dated the origin of human subfamilies using phylogenetic profiling, which enlarged the Rab repertoire of the Last Eukaryotic Common Ancestor with Rab14, 32 and RabL4. Furthermore, a detailed analysis of the Choanoflagellate Monosiga brevicollis Rab family pinpointed the changes that accompanied the emergence of Metazoan multicellularity, mainly an important expansion and specialisation of the secretory pathway. Lastly, we experimentally establish tissue specificity in expression of mouse Rabs and show that neo-functionalisation best explains the emergence of new human Rab subfamilies. With the Rabifier and RabDB, we provide tools that easily allows non-bioinformaticians to integrate thousands of Rabs in their analyses. RabDB is designed to enable the cell biology community to keep pace with the increasing number of fully-sequenced genomes and change the scale at which we perform comparative analysis in cell biology.
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