Guanine Triphosphate Research Articles

Identification of a guanine-specific pocket in the protein N of SARS-CoV-2

The SARS-CoV-2 nucleocapsid protein (N) is responsible for RNA binding. Here we report the crystal structure of the C-terminal domain (NCTD) in open and closed conformations and in complex with guanine triphosphate, GTP. The crystal structure and biochemical studies reveal a specific interaction between the guanine, a nucleotide enriched in the packaging signals regions of coronaviruses, and a highly conserved tryptophan residue (W330). In addition, EMSA assays with SARS-CoV-2 derived RNA hairpin loops from a putative viral packaging sequence showed the preference interaction of the N-CTD to RNA oligonucleotides containing G and the loss of the specificity in the mutant W330A. Here we propose that this interaction may facilitate the viral assembly process. In summary, we have identified a specific guanine-binding pocket in the N protein that may be used to design viral assembly inhibitors.

The structure–function analysis of Obg‐like GTPase proteins along the evolutionary tree from bacteria to humans

Obg proteins belong to P‐loop guanine triphosphatase (GTPase) that are conserved from bacteria to humans. Like other GTPases, Obg cycles between guanine triphosphate (GTP) bound “on” state and guanine diphosphate (GDP)‐bound “off” state, thereby controlling various cellular processes. Different members of this group have unique structural characteristics; a conserved glycine‐rich N‐terminal domain known as obg fold, a central conserved nucleotide binding domain, and a less conserved C‐terminal domain of other functions. Obg is a ribosome dependent GTPase helps in ribosome maturation by interacting with several proteins of the 50S subunit of the ribosome. Obg proteins have been widely considered as a regulator of cellular functions, helping in DNA replication, cell division. Apart from that, this protein also takes part in various stress adaptation pathways like a stringent response, sporulation, and general stress response. In this particular review, the structural features of ObgE have been highlighted and how the structure plays important role in interacting with regulators like GTP, ppGpp that are crucial for executing biological function has been orchestrated. In particular, we believe that Obg‐like proteins can provide a link between different global pathways that are necessary for fine‐tuning cellular processes to maintain the cellular energy status.

CAR Co-Operates With Integrins to Promote Lung Cancer Cell Adhesion and Invasion.

The coxsackie and adenovirus receptor (CAR) is a member of the junctional adhesion molecule (JAM) family of adhesion receptors and is localised to epithelial cell tight and adherens junctions. CAR has been shown to be highly expressed in lung cancer where it is proposed to promote tumor growth and regulate epithelial mesenchymal transition (EMT), however the potential role of CAR in lung cancer metastasis remains poorly understood. To better understand the role of this receptor in tumor progression, we manipulated CAR expression in both epithelial-like and mesenchymal-like lung cancer cells. In both cases, CAR overexpression promoted tumor growth in vivo in immunocompetent mice and increased cell adhesion in the lung after intravenous injection without altering the EMT properties of each cell line. Overexpression of WTCAR resulted in increased invasion in 3D models and enhanced β1 integrin activity in both cell lines, and this was dependent on phosphorylation of the CAR cytoplasmic tail. Furthermore, phosphorylation of CAR was enhanced by substrate stiffness in vitro, and CAR expression increased at the boundary of solid tumors in vivo. Moreover, CAR formed a complex with the focal adhesion proteins Src, Focal Adhesion Kinase (FAK) and paxillin and promoted activation of the Guanine Triphosphate (GTP)-ase Ras-related Protein 1 (Rap1), which in turn mediated enhanced integrin activation. Taken together, our data demonstrate that CAR contributes to lung cancer metastasis via promotion of cell-matrix adhesion, providing new insight into co-operation between cell-cell and cell-matrix proteins that regulate different steps of tumorigenesis.

Outstanding Enhancement of Curcumin Fluorescence in PDDA and Silica Nanoparticles Coated DMPC Liposomes Based Nanocapsules: Application for Selective Estimation of ATP**

AbstractHere we illustrate the synthesis of, 2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC) liposome based poly(dimethyldiallylammonium chloride) (PDDA) nanocapsule where PDDA and silica nanoparticles are coated on the surface of curcumin embedded liposomes. The prepared nanocapsule was analyzed using UV‐vis spectroscopy, fluorescence spectroscopy, Dynamic Light Scattering technique, Scanning Electron Microscopy, Zeta‐potential, Thermogravimetric analysis and X‐ray diffraction method. The fluorescence intensity of DMPC nanocapsules was found to be exceedingly, 60‐fold, higher than free curcumin, thus, it boosts potential of such nanocapsules for various applications. The detection of Adinosine Triphosphate (ATP) was carried out by monitoring the change in the fluorescence emission of this DMPC nanocapsules while increasing the concentration of ATP molecule in the tested sample. Interestingly, fluorescence of free curcumin did not respond to ATP, whereas the fluorescence intensity of nanocapsules increased by the increase of ATP concentration, which establishes that materials prepared using nanotechnological modification of probe molecule can selectively target a particular analytical species. The nanocapsules based probe gave a linear correlation between the fluorescence intensity of DMPC nanocapsules in the concentration range 1 μM −100 μM with the limit of detection equals to 0.5 μM. The described method is not interfered by uracil triphosphate, guanine triphosphate, cytosine triphosphate and thymine triphosphate molecules and showed good stability.

Identification and Characterization of Small‐Molecule Ras Modulators and their Impact in Cancer Drug Discovery

Ras is a small GTPase enzyme that binds guanine diphosphate (GDP), a nucleotide substrate, to its guanine‐nucleotide binding pocket. Ras activation occurs by the coordination of SOS, a guanine nucleotide exchange factor, which catalyzes the substitution of GDP for guanine triphosphate (GTP), rendering the Ras protein in an active state. Oncogenic Ras mutants are constitutively active, propagating proliferative cell signaling that leads to aberrant cell growth and metastatic tumors that are present in 30% of all human cancers. Discovery of a potent, small molecule Ras inhibitor has been challenging. We have characterized a novel class of Ras modulators that exhibit both Ras activation. These small molecules were discovered using Ras‐SOS molecular modeling and computational docking studies focused on identifying potential compound binding to the Ras‐SOS complex interface. Several in‐vitro biochemical assays were performed to elucidate the structural significance, binding kinetics, and nucleotide exchange effects of these small molecule binders. A fluorescence nucleotide exchange assay was optimized to discover compounds that had an atypical nucleotide exchange rate (off‐state to on‐state) in a kinetic assay. Compounds discovered in the assay were then analyzed by Surface Plasmon Resonance (SPR) to further elucidate details on the binding kinetics between Ras and compound as well as between Ras and SOS in the presence of the compound. Of the examined compounds, several exhibited activation of the Ras‐SOS catalytic reaction. An ongoing structure activity relationship (SAR) study has identified essential components that facilitate binding to the Ras:SOS interface. This presumably allosteric interaction may have uncovered an unprobed regulatory site that can be manipulated to create Ras inhibitors. This discovery of activator compounds provides a promising new direction for the development of potent Ras modulators.Support or Funding InformationThe Center for Healthcare Innovation (CHI), Stevens Institute of Technology

Ras and RTK: PI3K Activation, PIP3 Formation, Signal Transduction, Cancer Creation

Ras mutations are implicated in over 30% of all cancers. Under normal conditions, Ras becomes active by binding to guanine triphosphate (GTP) to stimulate cell growth and division and other cell pathways. When Ras acquires specific mutations, it no longer binds the GTPase‐activating protein (GAP), which impairs the GAP GTPase activity, trapping Ras in its active state and leading to uncontrolled cell proliferation. The Ras family of G‐proteins play a role in recruitment of phosphatidylinositol‐3‐kinase (PI3K) to the membrane and stimulation of PIP3 lipid signaling. The Ras family consists of 3 major isoforms: H‐Ras, K‐Ras, and N‐Ras; all having similar functions in different pathways. The primary sequence of amino acids among the Ras isoforms is highly conserved with differences existing primarily between their C‐terminal tails. The Falke lab is studying the H‐Ras variant. H‐Ras, in its active GTP‐bound state, and receptor tyrosine kinase (RTK) synergistically recruit PI3K to the membrane and stimulate PI3K kinase activity. During recruitment to the lipid bilayer, PI3K binds its substrate lipid phosphatidylinositol‐4‐5‐bisphosphate (PIP2) and also binds adenosine triphosphate (ATP) and then phosphorylates PIP2, creating phosphatidylinositol (3,4,5)‐trisphosphate (PIP3). PIP3 then plays a central role in many cell growth and regulatory pathways by recruiting to the membrane many proteins that bind to its headgroup. Specific surface amino acid sidechains on Ras that bind to specific surface amino acid sidechains on the Ras‐binding domain (RBD) of PI3K during their interaction to assemble the fully active PI3K complex were modeled. Drugs that inhibit Ras or PI3K or their interaction may be effective in attacking cancer cell growth. Ultimately, understanding Ras and the mechanism of PI3K activation promotes understanding of a large subset of human cancers and facilitates the development of targeted treatments. The Longmont High School SMART (Students Modeling A Research Topic) Team modeled wild‐type H‐Ras and oncogenic mutants using 3D printing technology to investigate structure‐function relationships.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases.

Small, monomeric guanine triphosphate hydrolases (GTPases) are ubiquitous cellular integrators of signaling. A signal activates the GTPase, which then binds to an effector molecule to relay a signal inside the cell. The GTPase effector trap flow cytometry assay (G-Trap) utilizes bead-based protein immobilization and dual-color flow cytometry to rapidly and quantitatively measure GTPase activity status in cell or tissue lysates. Beginning with commercial cytoplex bead sets that are color-coded with graded fluorescence intensities of a red (700 nm) wavelength, the bead sets are derivatized to display glutathione on the surface through a detailed protocol described here. A different glutathione-S-transferase-effector protein (GST-effector protein) can then be attached to the surface of each set. For the assay, users can incubate bead sets individually or in a multiplex format with lysates for rapid, selective capture of active, GTP-bound GTPases from a single sample. After that, flow cytometry is used to identify the bead-borne GTPase based on red bead intensity, and the amount of active GTPase per bead is detected using monoclonal antibodies conjugated to a green fluorophore or via labeled secondary antibodies. Three examples are provided to illustrate the efficacy of the effector-functionalized beads for measuring the activation of at least five GTPases in a single lysate from fewer than 50,000 cells.

Dynamic Role of the GTP Energy Metabolism in Cancers.

Rapid growing cells like tumor cells need a vast amount of energy to match their high metabolic demand. Guanine triphosphate (GTP) is one of major cellular metabolites and served as a building block for RNA and DNA as well as an energy source to drive cellular activities such as intracellular trafficking, the cell migration and translation. However, how cancer cells regulate GTP energy levels to adapt for their high demand remain largely unknown yet. In addition, how cells detect GTP levels remains unknown. In this seminar, I will introduce our recent findings that uncover dramatic change of GTP metabolism in cancer cells and a GTP sensing kinase that regulate metabolism for tumorigenesis.(Presented at the 1918th Meeting, March 3, 2016).

Changes in the GEF-H1 Pathways after Traumatic Brain Injury

Brains undergo significant remodeling after traumatic brain injury (TBI). The Rho guanine triphosphate (GTP)ase pathways control brain remodeling during development and under pathological conditions. How the Rho GTPase pathways are regulated in the brain after TBI remains largely unknown, however. This study used the rat fluid percussion injury model to investigate changes in the Rho GTPase pathways after TBI. The results showed that TBI leads to activation and translocation of RhoA and Rac1 proteins from cytosolic fraction to the membrane fraction after injury. Consistently, the Rho guanine nucleotide exchange factors GEF-H1 and Cool-2/αPix are significantly activated by dephosphorylation and accumulation in the cytosolic fractions during the post-TBI phase. Because the Rho GTPase pathways are key regulators of brain remodeling, these results depict regulatory mechanisms of the Rho GTPase pathways after TBI, and pave the way for the study of therapeutic agents targeting the Rho GTPase pathways for functional recovery after TBI.

Regulated proteolysis of Candida albicans Ras1 is involved in morphogenesis and quorum sensing regulation

In Candida albicans, a fungal pathogen, the small G-protein Ras1 regulates many important behaviors including white-opaque switching, biofilm formation, and the induction and maintenance of hyphal growth. Like other Ras proteins, Ras1 is activated upon guanine triphosphate binding, and its activity is further modulated by post-translational lipid modifications. Here, we report that the levels of membrane-associated, full-length Ras1 were higher in hyphae than in yeast, and that yeast contained a shorter, soluble Ras1 species that resulted from cleavage. Deletion of the putative cleavage site led to more rapid induction of hyphal growth and delayed hypha-to-yeast transitions. The cleaved Ras1 species was less able to activate its effector, adenylate cyclase (Cyr1), unless tethered to the membrane by a heterologous membrane-targeting domain. Ras1 cleavage was repressed by cAMP-signalling, indicating the presence of a positive feedback loop in which Cyr1 and cAMP influence Ras1. The C. albicans quorum sensing molecule farnesol, which inhibits Cyr1 and represses filamentation, caused an increase in the fraction of Ras1 in the cleaved form, particularly in nascent yeast formed from hyphae. This newly recognized mode of Ras regulation may control C. albicans Ras1 activity in important ways.

Guanine Triphosphate–Cyclohydrolase 1–Deficient Dopa-Responsive Dystonia Presenting as Frequent Falling in 2 Children

Guanine triphosphate (GTP)-cyclohydrolase 1 (GCH1)-deficient dopa-responsive dystonia is caused by GCH1 gene mutation. Two children presenting with frequent daily falling are reported with GCH1 gene mutations with persistent response to low-dose levodopa/carbidopa. Typical and atypical clinical features associated with GCH1 mutations are also reviewed.

Molecular dynamics simulations reveal structural coordination of Ffh‐FtsY heterodimer toward GTPase activation

Two homologous GTPases (guanine-triphosphatases) in the signal recognition particle (SRP) and its receptor (SR) use their cumulative energy during GTP (guanine-triphosphate) hydrolysis to control the co-translational protein targeting process. Distinct from classical GTPases, which rely on external factors to hydrolyze GTP, SRP GTPases stimulate one another's activity in a self-sufficient manner upon SRP-SR complex association. Although both ground-state and putative transition-state GTP analogs have been used to recapitulate the state of GTPase activation, the underlying mechanism of the activated state still remains elusive. In particular, several residues that were placed in pending positions have been shown to be important to GTP hydrolysis in biochemical studies. Here, we examined the stability and dynamics of three interaction networks involving these residues and discovered that they contribute to the GTPase activation via well-tuned conformational changes. The crystallographically identified pending residues Ffh:R191/FtsY:R195 undergo extensive conformational rearrangements to form persisted interactions with FtsY:E284/Ffh:E274, explaining the biochemically observed defective effect of R191 mutant to the activation of both GTPases. In addition, the side chain of FtsY:R142, one of the most important catalytic residues, rotates to an extended conformation that could more efficiently maintain the electrostatic balance for GTP hydrolysis. Finally, the invariant residues Ffh:G190 and FtsY:G194, instead of the supposed auxiliary water molecules, are proposed to stabilize the nucleophilic waters during GTPase activation. In complementary to experimental observations, these findings suggest a more favorable interaction model for SRP GTPase activation and would thus benefit to our understanding of how SRP GTPases regulate the protein targeting pathway.

The Crystal Structure of the Signal Recognition Particle in Complex with Its Receptor

Cotranslational targeting of membrane and secretory proteins is mediated by the universally conserved signal recognition particle (SRP). Together with its receptor (SR), SRP mediates the guanine triphosphate (GTP)-dependent delivery of translating ribosomes bearing signal sequences to translocons on the target membrane. Here, we present the crystal structure of the SRP:SR complex at 3.9 angstrom resolution and biochemical data revealing that the activated SRP:SR guanine triphosphatase (GTPase) complex binds the distal end of the SRP hairpin RNA where GTP hydrolysis is stimulated. Combined with previous findings, these results suggest that the SRP:SR GTPase complex initially assembles at the tetraloop end of the SRP RNA and then relocalizes to the opposite end of the RNA. This rearrangement provides a mechanism for coupling GTP hydrolysis to the handover of cargo to the translocon.

Activation of Rho GTPases by DOCK Exchange Factors Is Mediated by a Nucleotide Sensor

Activation of Rho guanosine triphosphatases (GTPases) to the guanine triphosphate (GTP)-bound state is a critical event in their regulation of the cytoskeleton and cell signaling. Members of the DOCK family of guanine nucleotide exchange factors (GEFs) are important activators of Rho GTPases, but the mechanism of activation by their catalytic DHR2 domain is unknown. Through structural analysis of DOCK9-Cdc42 complexes, we identify a nucleotide sensor within the alpha10 helix of the DHR2 domain that contributes to release of guanine diphosphate (GDP) and then to discharge of the activated GTP-bound Cdc42. Magnesium exclusion, a critical factor in promoting GDP release, is mediated by a conserved valine residue within this sensor, whereas binding of GTP-Mg2+ to the nucleotide-free complex results in magnesium-inducing displacement of the sensor to stimulate discharge of Cdc42-GTP. These studies identify an unusual mechanism of GDP release and define the complete GEF catalytic cycle from GDP dissociation followed by GTP binding and discharge of the activated GTPase.

RAS in cellular transformation and senescence

In 1964, a transforming retrovirus was described that produced tumours in mice [1]. The Harvey rat sarcoma virus, aptly named H-RAS after the discovering scientist, encoded an oncogene that had been hijacked from its host. A similar virus was isolated in 1970 and was named the Kirsten rat sarcoma virus or K-RAS [2]. In 1982, the human genes homologous to the viral genes were elucidated and were designated c-H-RAS and c-K-RAS [3], and subsequently the third and final RAS family member was isolated from human neuroblastoma samples and termed c-N-RAS. An examination of other human cancers also revealed RAS genes capable of transforming mammalian cells, and interestingly, these alleles were transforming due to point mutations that inactivated the proteins’ intrinsic guanine triphosphatase (GTPase) activity [4,5]. RAS functions downstream of mitogenic growth factor receptors. Following the ligation with growth factors, these receptor tyrosine kinases undergo autophosphorylation and thereby recruit adaptor proteins that bind guanine nucleotide exchange factors (GEFs) (for review, see [6]). GEFs catalyse the exchange of guanine diphosphate (GDP) for guanine triphosphate (GTP) on small GTP-binding proteins, including RAS. GTP-bound RAS subsequently interacts with a number of effectors to regulate cellular proliferation and survival, notably including the RAF family of proteins and type I phosphoinositol-3-kinase (PI3K). RAS proteins mutated at codons 12, 13, or 61 are rendered constitutively GTP bound and consequently have increased affinity to RAF proteins and PI3K, leading to activation of downstream pathways. The RAF family of serine/threonine kinases consists of three members: ARAF, BRAF, and CRAF [6]. Following activation by RAS, RAF kinases phosphorylate Mitogen-Activated Protein Kinase (MEK) kinases (MEK1 and MEK2), resulting in their activation. MEK proteins subsequently phosphorylate Extracellular Regulated MAP Kinase (ERK) (ERK1 and ERK2) in the cytoplasm, resulting in their activation and nuclear translocation. ERK kinases have a variety of cytoplasmic and nuclear targets, importantly including ETS transcription factors to regulate the expression of many pro-proliferative genes. RAS-GTP also interacts with and stimulates the activity of PI3K [6]. Upon activation, PI3K catalyses the conversion of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) to produce phosphatidylinositol-3,4,5trisphosphate (PtdIns(3,4,5)P3). Increased local concentrations of PtdIns(3,4,5)P3 result in recruitment of the kinase PDK1 via its pleckstrin homology domain, which phosphorylates and activates the kinase AKT. AKT in turn promotes cellular survival and activates mammalian target of rapamycin (mTOR) to increase ribosomal biogenesis and messenger RNA (mRNA) translation. Additionally, active PI3K also stimulates the GTP-binding protein RAC, a regulator of the actin cytoskeleton. Therefore, rather than relying upon a single pathway for cellular transformation, it is hypothesised that numerous alterations in multiple biochemical pathways collectively promote cellular transformation by oncogenic RAS. Oncogenic RAS mutations are the most common oncogenic events identified in human tumours, being found in 30% of human cancers. KRAS is the most commonly mutated member of the RAS family, present in over 90% of ductal pancreatic cancers, 40−50% colorectal cancers, and 30% of non-small cell lung cancers (6). A detailed characterisation of the cellular and molecular pathways altered by oncogenic RAS has been pursued in order to identify potential therapeutic targets for such cancers, but despite its expected role in promoting tumourigenesis, ectopic expression of oncogenic RAS in primary cell cultures induces a paradoxical irreversible growth arrest known as oncogene-induced senescence (OIS) [7]. OIS is characterised by a flattened cellular morphology, a large nucleus with a prominent nucleolus, chromatin reorganisation, and activation of the p53 and p16INK4a pathways [7]. Although ectopic expression of oncogenic RAS in primary cells induces OIS, the exact molecular mechanisms remain unclear. Studies have

Current evidence for a modulation of low back pain by human genetic variants

The manifestation of chronic back pain depends on structural, psychosocial, occupational and genetic influences. Heritability estimates for back pain range from 30% to 45%. Genetic influences are caused by genes affecting intervertebral disc degeneration or the immune response and genes involved in pain perception, signalling and psychological processing. This inter-individual variability which is partly due to genetic differences would require an individualized pain management to prevent the transition from acute to chronic back pain or improve the outcome. The genetic profile may help to define patients at high risk for chronic pain. We summarize genetic factors that (i) impact on intervertebral disc stability, namely Collagen IX, COL9A3, COL11A1, COL11A2, COL1A1, aggrecan (AGAN), cartilage intermediate layer protein, vitamin D receptor, metalloproteinsase-3 (MMP3), MMP9, and thrombospondin-2, (ii) modify inflammation, namely interleukin-1 (IL-1) locus genes and IL-6 and (iii) and pain signalling namely guanine triphosphate (GTP) cyclohydrolase 1, catechol-O-methyltransferase, mu opioid receptor (OPMR1), melanocortin 1 receptor (MC1R), transient receptor potential channel A1 and fatty acid amide hydrolase and analgesic drug metabolism (cytochrome P450 [CYP]2D6, CYP2C9).

Characterization of Mutant MUTYH Proteins Associated With Familial Colorectal Cancer

The human mutyh gene encodes a base excision repair protein that prevents G:C to T:A transversions in DNA. Biallelic mutations in this gene are associated with recessively inherited familial colorectal cancer. The aim of this study was to characterize the functional activity of mutant-MUTYH and single-nucleotide polymorphism (SNP)-MUTYH proteins involving familial colorectal cancer. MUTYH variants were cloned and assayed for their glycosylase and DNA binding activities using synthetic double-stranded oligonucleotide substrates by analyzing cleavage products by polyacrylamide gel electrophoresis. In this study, we have characterized 9 missense/frameshift mutants and 2 SNPs for their DNA binding and repair activity in vitro. Two missense mutants (R260Q and G382D) were found to be partially active in both glycosylase and DNA binding, whereas 3 other missense mutants (Y165C, R231H, and P281L) were severely defective in both activities. All of the frameshift mutants (Y90X, Q377X, E466X, and 1103delC) were completely devoid of both glycosylase and DNA binding activities. One SNP (V22M) showed the same activity as wild-type MUTYH protein, but the other SNP (Q324H) was partially impaired in adenine removal. This study of MUTYH mutants suggests that certain SNPs may be as partially dysfunctional in base excision repair as missense-MUTYH mutants and lead to colorectal carcinogenesis.

Coactivation of G Protein Signaling by Cell-Surface Receptors and an Intracellular Exchange Factor

G protein-coupled receptors (GPCRs) mediate responses to a broad range of chemical and environmental signals. In yeast, a pheromone-binding GPCR triggers events leading to the fusion of haploid cells. In general, GPCRs function as guanine-nucleotide exchange factors (GEFs); upon agonist binding, the receptor induces a conformational change in the G protein alpha subunit, resulting in exchange of guanine diphosphate (GDP) for guanine triphosphate (GTP) and in signal initiation. Signaling is terminated when GTP is hydrolyzed to GDP [1]. This well-established paradigm has in recent years been revised to include new components that rates of GDP release, GTP binding [2-8], and GTP hydrolysis[9, 10]. Here we report the discovery of a nonreceptor GEF, Arr4. Like receptors, Arr4 binds directly to the G protein,accelerates guanine-nucleotide exchange, and stabilizes the nucleotide-free state of the a subunit. Moreover, Arr4 promotes G protein-dependent cellular responses, including mitogen-activated protein kinase (MAPK) phosphorylation,new-gene transcription, and mating. In contrast to knownGPCRs, however, Arr4 is not a transmembrane receptor,but rather a soluble intracellular protein. Our data suggest that intracellular proteins function in cooperation with mating pheromones to amplify G protein signaling, thereby leading to full pathway activation.

Mutated D4-guanine diphosphate–dissociation inhibitor is found in human leukemic cells and promotes leukemic cell invasion

Rho GTPase may be involved in human cancer invasion via the augmentation of cell motility and adhesion. We report on two point mutations of the D4-guanine diphosphate (GDP)-dissociation inhibitor (GDI) gene, one of the Rho-GDIs, which were found in a human leukemic cell line, Reh, and the mutated D4-GDI functions as an accelerator of leukemic cell invasion. We investigated the altered activity of GDP dissociation by mutated (mt) D4-GDI and the functions of this mt and wild-type (wt) D4-GDI in invasion. The mice inoculated with wt or mt D4-GDI vector-transfected Raji cells were observed and examined pathologically. Adhesiveness and cell motility of wt or mt D4-GDI vector-transfected Raji cells were examined. Finally, it was examined whether Rho activation was changed by mutation of D4-GDI under the condition of Rho-GDI knockdown. Two point mutations of the D4-GDI gene were found in Reh cells. The region of mutations is conserved among members of the Rho-GDI family at the amino acid level. D4-GDI with two mutations (V68L and V69A) functioned in a dominant negative manner in the inhibition of GDP dissociation from Rho. Severe combined immune-deficient mice inoculated with Raji cells developed hemiparalysis. The Raji cells were present in bone marrow and peripheral blood, and hepatic invasion was observed in 20% of the mice. Mice inoculated with wt D4-GDI vector-transfected Raji cells (wt D4) showed later paralysis and none developed hepatic invasion. Mice inoculated with mt D4-GDI-transfected Raji cells (mt D4) showed a 5-day reduction in the time to paraplegia and death. In addition, hepatic invasion was evident in 80% of mice transplanted with mt D4 cells. There were no differences in growth rates and amounts of guanine triphosphate (GTP)-bound Rho, cdc42, or Rac among all clones, however, GTP-bound Rho in mt D4 clone with short hairpin RNA (shRNA) vector for Rho-GDI knockdown was increased compared with wt D4 clone with shRNA vector for Rho-GDI knockdown. The mt D4 cells showed an augmentation of adhesiveness and cell motility. On the other hand, wt D4 cells showed a decreased ability of cell motility. These results suggest the mutated D4-GDI functions as a dominant negative molecule against the wt D4-GDI and accelerates invasion via regulation of cytoskeletal machinery.

Biochemical Studies on the Mechanism of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Resistance to 1-(β-d-Dioxolane)Thymine Triphosphate

A large panel of drug-resistant mutants of human immunodeficiency virus type 1 reverse transcriptase (RT) was used to study the mechanisms of resistance to 1-(beta-d-dioxolane)thymine triphosphate (DOT-TP) and other nucleotide analogs. RT containing thymidine analog-associated mutations (TAM) or RT with a T69S-SG insertion in combination with TAM removed 3'-azido-3'-deoxythymidine-5'-monophosphate or tenofovir more efficiently than DOT-monophosphate from chain-terminated DNA primer/template through ATP-mediated pyrophosphorolysis. For non-ATP-dependent discrimination toward DOT-TP, high levels of resistance were found for RT bearing the Q151M mutation with family mutations, while RT bearing only the M184V or the Y115F mutation conferred no resistance to DOT-TP. A lower degree of resistance to DOT-TP than to tenofovir diphosphate or carbovir-TP was found for RT containing the K65R mutation. In the present studies, 1-(beta-d-dioxolane)guanine triphosphate, another nucleotide with a dioxolane sugar moiety, showed a resistance profile similar to that of DOT-TP. The results suggest that DOT, compared with other approved nucleoside analogs, is overall more resilient to mutations such as TAM, M184V, and K65R, which are commonly found in viruses derived from subjects failing multinucleoside therapy.