RhoG Protein Regulates Platelet Granule Secretion and Thrombus Formation in Mice

Rho GTPases such as Rac, RhoA, and Cdc42 are vital for normal platelet function, but the role of RhoG in platelets has not been studied. In other cells, RhoG orchestrates processes integral to platelet function, including actin cytoskeletal rearrangement and membrane trafficking. We therefore hypothesized that RhoG would play a critical role in platelets. Here, we show that RhoG is expressed in human and mouse platelets and is activated by both collagen-related peptide (CRP) and thrombin stimulation. We used RhoG^−/− mice to study the function of RhoG in platelets. Integrin activation and aggregation were reduced in RhoG^−/− platelets stimulated by CRP, but responses to thrombin were normal. The central defect in RhoG^−/− platelets was reduced secretion from α-granules, dense granules, and lysosomes following CRP stimulation. The integrin activation and aggregation defects could be rescued by ADP co-stimulation, indicating that they are a consequence of diminished dense granule secretion. Defective dense granule secretion in RhoG^−/− platelets limited recruitment of additional platelets to growing thrombi in flowing blood in vitro and translated into reduced thrombus formation in vivo. Interestingly, tail bleeding times were normal in RhoG^−/− mice, suggesting that the functions of RhoG in platelets are particularly relevant to thrombotic disorders.     

Characterization of Anopheles gambiae Transglutaminase 3 (AgTG3) and Its Native Substrate Plugin

Male Anopheles mosquitoes coagulate their seminal fluids via cross-linking of a substrate, called Plugin, by the seminal transglutaminase AgTG3. Formation of the “mating plug” by cross-linking Plugin is necessary for efficient sperm storage by females. AgTG3 has a similar degree of sequence identity (∼30%) to both human Factor XIII (FXIII) and tissue transglutaminase 2 (hTG2). Here we report the solution structure and in vitro activity for the cross-linking reaction of AgTG3 and Plugin. AgTG3 is a dimer in solution and exhibits Ca²⁺-dependent nonproteolytic activation analogous to cytoplasmic FXIII. The C-terminal domain of Plugin is predominantly α-helical with extended tertiary structure and oligomerizes in solution. The specific activity of AgTG3 was measured as 4.25 × 10⁻² units mg⁻¹. AgTG3 is less active than hTG2 assayed using the general substrate TVQQEL but has 8–10× higher relative activity when Plugin is the substrate. Mass spectrometric analysis of cross-linked Plugin detects specific peptides including a predicted consensus motif for cross-linking by AgTG3. These results support the development of AgTG3 inhibitors as specific and effective chemosterilants for A. gambiae.     

Hematopoietic-specific Rho GTPases Rac2 and RhoH and human blood disorders

The small guanosine triphosphotases (GTPases) Rho proteins are members of the Ras-like superfamily. Similar to Ras, most Rho GTPases cycle between active GTP-bound, and inactive GDP-bound conformations and act as molecular switches that control multiple cellular functions. While most Rho GTPases are expressed widely, the expression of Rac2 and RhoH are restricted to hematopoietic cells. RhoH is an atypical GTPase that lacks GTPase activity and remains in the active conformation. The generation of mouse knock-out lines has led to new understanding of the functions of both of these proteins in blood cells. The phenotype of these mice also led to the identification of mutations in human RAC2 and RHOH genes and the role of these proteins in immunodeficiency diseases. This review outlines the basic biology of Rho GTPases, focusing on Rac and RhoH and summarizes human diseases associated with mutations of these genes.     

Protein Degradation and Quality Control in Cells from Laforin and Malin Knockout Mice

Lafora disease is a progressive myoclonus epilepsy caused by mutations in the EPM2A or EPM2B genes that encode a glycogen phosphatase, laforin, and an E3 ubiquitin ligase, malin, respectively. Lafora disease is characterized by accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle, heart, and liver. The laforin-malin complex has been proposed to play a role in the regulation of glycogen metabolism and protein quality control. We evaluated three arms of the protein degradation/quality control process (the autophago-lysosomal pathway, the ubiquitin-proteasomal pathway, and the endoplasmic reticulum (ER) stress response) in mouse embryonic fibroblasts from Epm2a^−/−, Epm2b^−/−, and Epm2a^−/− Epm2b^−/− mice. The levels of LC3-II, a marker of autophagy, were decreased in all knock-out cells as compared with wild type even though they still showed a slight response to starvation and rapamycin. Furthermore, ribosomal protein S6 kinase and S6 phosphorylation were increased. Under basal conditions there was no effect on the levels of ubiquitinated proteins in the knock-out cells, but ubiquitinated protein degradation was decreased during starvation or stress. Lack of malin (Epm2b^−/− and Epm2a^−/− Epm2b^−/− cells) but not laforin (Epm2a^−/− cells) decreased LAMP1, a lysosomal marker. CHOP expression was similar in wild type and knock-out cells under basal conditions or with ER stress-inducing agents. In conclusion, both laforin and malin knock-out cells display mTOR-dependent autophagy defects and reduced proteasomal activity but no defects in the ER stress response. We speculate that these defects may be secondary to glycogen overaccumulation. This study also suggests a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal.     

Structural Insights into the Activation of the RhoA GTPase by the Lymphoid Blast Crisis (Lbc) Oncoprotein

The small GTPase RhoA promotes deregulated signaling upon interaction with lymphoid blast crisis (Lbc), the oncogenic form of A-kinase anchoring protein 13 (AKAP13). The onco-Lbc protein is a hyperactive Rho-specific guanine nucleotide exchange factor (GEF), but its structural mechanism has not been reported despite its involvement in cardiac hypertrophy and cancer causation. The pleckstrin homology (PH) domain of Lbc is located at the C-terminal end of the protein and is shown here to specifically recognize activated RhoA rather than lipids. The isolated dbl homology (DH) domain can function as an independent activator with an enhanced activity. However, the DH domain normally does not act as a solitary Lbc interface with RhoA-GDP. Instead it is negatively controlled by the PH domain. In particular, the DH helical bundle is coupled to the structurally dependent PH domain through a helical linker, which reduces its activity. Together the two domains form a rigid scaffold in solution as evidenced by small angle x-ray scattering and ¹H,¹³C,¹⁵N-based NMR spectroscopy. The two domains assume a “chair” shape with its back possessing independent GEF activity and the PH domain providing a broad seat for RhoA-GTP docking rather than membrane recognition. This provides structural and dynamical insights into how DH and PH domains work together in solution to support regulated RhoA activity. Mutational analysis supports the bifunctional PH domain mediation of DH-RhoA interactions and explains why the tandem domain is required for controlled GEF signaling.     

Establishment and Characterization of PCL12, a Novel CD5+ Chronic Lymphocytic Leukaemia Cell Line

Immortalized cell lines representative of chronic lymphocytic leukemia (CLL) can assist in understanding disease pathogenesis and testing new therapeutic agents. At present, very few representative cell lines are available. We here describe the characterization of a new cell line (PCL12) that grew spontaneously from the peripheral blood (PB) of a CLL patient with progressive disease and EBV infection. The CLL cell origin of PCL12 was confirmed after the alignment of its IGH sequence against the “original” clonotypic sequence. The IGH gene rearrangement was truly unmutated and no CLL-related cytogenetic or genetic lesions were detected. PCL12 cells express CD19, CD20, CD5, CD23, low levels of IgM and IgD and the poor-outcome-associated prognostic markers CD38, ZAP70 and TCL1. In accordance with its aggressive phenotype the cell line is inactive in terms of LYN and HS1 phosphorylation. BcR signalling pathway is constitutively active and anergic in terms of p-ERK and Calcium flux response to α-IgM stimulation. PCL12 cells strongly migrate in vitro in response to SDF-1 and form clusters. Finally, they grow rapidly and localize in all lymphoid organs when xenotrasplanted in Rag2^-/-γc^-/- mice. PCL12 represents a suitable preclinical model for testing pharmacological agents.     

Double Strand Break Unwinding and Resection by the Mycobacterial Helicase-Nuclease AdnAB in the Presence of Single Strand DNA-binding Protein (SSB)

Mycobacterial AdnAB is a heterodimeric DNA helicase-nuclease and 3′ to 5′ DNA translocase implicated in the repair of double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Inclusion of mycobacterial single strand DNA-binding protein (SSB) in reactions containing linear plasmid dsDNA allowed us to study the AdnAB helicase under conditions in which the unwound single strands are coated by SSB and thereby prevented from reannealing or promoting ongoing ATP hydrolysis. We found that the AdnAB motor catalyzed processive unwinding of 2.7–11.2-kbp linear duplex DNAs at a rate of ∼250 bp s⁻¹, while hydrolyzing ∼5 ATPs per bp unwound. Crippling the AdnA phosphohydrolase active site did not affect the rate of unwinding but lowered energy consumption slightly, to ∼4.2 ATPs bp⁻¹. Mutation of the AdnB phosphohydrolase abolished duplex unwinding, consistent with a model in which the “leading” AdnB motor propagates a Y-fork by translocation along the 3′ DNA strand, ahead of the “lagging” AdnA motor domain. By tracking the resection of the 5′ and 3′ strands at the DSB ends, we illuminated a division of labor among the AdnA and AdnB nuclease modules during dsDNA unwinding, whereby the AdnA nuclease processes the unwound 5′ strand to liberate a short oligonucleotide product, and the AdnB nuclease incises the 3′ strand on which the motor translocates. These results extend our understanding of presynaptic DSB processing by AdnAB and engender instructive comparisons with the RecBCD and AddAB clades of bacterial helicase-nuclease machines.     

Calpain 8/nCL-2 and Calpain 9/nCL-4 Constitute an Active Protease Complex,G-Calpain,Involved in Gastric Mucosal Defense

Calpains constitute a superfamily of Ca²⁺-dependent cysteine proteases, indispensable for various cellular processes. Among the 15 mammalian calpains, calpain 8/nCL-2 and calpain 9/nCL-4 are predominantly expressed in the gastrointestinal tract and are restricted to the gastric surface mucus (pit) cells in the stomach. Possible functions reported for calpain 8 are in vesicle trafficking between ER and Golgi, and calpain 9 are implicated in suppressing tumorigenesis. These highlight that calpains 8 and 9 are regulated differently from each other and from conventional calpains and, thus, have potentially important, specific functions in the gastrointestinal tract. However, there is no direct evidence implicating calpain 8 or 9 in human disease, and their properties and physiological functions are currently unknown. To address their physiological roles, we analyzed mice with mutations in the genes for these calpains, Capn8 and Capn9. Capn8^−/− and Capn9^−/− mice were fertile, and their gastric mucosae appeared normal. However, both mice were susceptible to gastric mucosal injury induced by ethanol administration. Moreover, the Capn8^−/− stomach showed significant decreases in both calpains 9 and 8, and the same was true for Capn9^−/−. Consistent with this finding, in the wild-type stomach, calpains 8 and 9 formed a complex we termed “G-calpain,” in which both were essential for activity. This is the first example of a “hybrid” calpain complex. To address the physiological relevance of the calpain 8 proteolytic activity, we generated calpain 8:C105S “knock-in” (Capn8^CS/CS) mice, which expressed a proteolytically inactive, but structurally intact, calpain 8. Although, unlike the Capn8^−/− stomach, that of the Capn8^CS/CS mice expressed a stable and active calpain 9, the mice were susceptible to ethanol-induced gastric injury. These results provide the first evidence that both of the gastrointestinal-tract-specific calpains are essential for gastric mucosal defense, and they point to G-calpain as a potential target for gastropathies caused by external stresses.     

Rheb (Ras Homologue Enriched in Brain)-dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Becomes Indispensable for Cardiac Hypertrophic Growth after Early Postnatal Period

Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb^−/−). Rheb^−/− mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb^−/− was lower than that in the control (Rheb^+/+) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb^+/+ mice but not in Rheb^−/− mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb^−/− hearts during the neonatal period. Rheb^−/− hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb^−/− hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb^−/− mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.     

Potentiation of Ligand Binding through Cooperative Effects in Monoamine Oxidase B

Crystallographic and biochemical studies have been employed to identify the binding site and mechanism for potentiation of imidazoline binding in human monoamine oxidase B (MAO B). 2-(2-Benzofuranyl)-2-imidazoline (2-BFI) inhibits recombinant human MAO B with a K_i of 8.3 ± 0.6 μm, whereas tranylcypromine-inhibited MAO B binds 2-BFI with a K_d of 9 ± 2 nm, representing an increase in binding energy Δ(ΔG) of −3.9 kcal/mol. Crystal structures show the imidazoline ligand bound in a site that is distinct from the substrate-binding cavity. Contributions to account for the increase in binding affinity upon tranylcypromine inhibition include a conformational change in the side chain of Gln²⁰⁶ and a “closed conformation” of the side chain of Ile¹⁹⁹, forming a hydrophobic “sandwich” with the side chain of Ile³¹⁶ on each face of the benzofuran ring of 2-BFI. Data with the I199A mutant of human MAO B and failure to observe a similar binding potentiation with rat MAO B, where Ile³¹⁶ is replaced with a Val residue, support an allosteric mechanism where the increased binding affinity of 2-BFI results from a cooperative increase in H-bond strength through formation of a more hydrophobic milieu. These insights should prove valuable in the design of high affinity and specific reversible MAO B inhibitors.     

Amyloid Precursor Protein (APP)/APP-like Protein 2 (APLP2) Expression Is Required to Initiate Endosome-Nucleus-Autophagosome Trafficking of Glypican-1-derived Heparan Sulfate

Anhydromannose (anMan)-containing heparan sulfate (HS) derived from the proteoglycan glypican-1 is generated in endosomes by an endogenously or ascorbate-induced S-nitrosothiol-catalyzed reaction. Processing of the amyloid precursor protein (APP) and APP-like protein 2 (APLP2) by β- and γ-secretases into amyloid β (Aβ) and Aβ-like peptides also takes place in these compartments. Moreover, anMan-containing HS suppresses the formation of toxic Aβ assemblies in vitro. We showed by using deconvolution immunofluorescence microscopy with an anMan-specific monoclonal antibody as well as ³⁵S labeling experiments that expression of APP/APLP2 is required for ascorbate-induced transport of HS from endosomes to the nucleus. Nuclear translocation was observed in wild-type mouse embryonic fibroblasts (WT MEFs), Tg2576 MEFs, and N2a neuroblastoma cells but not in APP^−/− and APLP2^−/− MEFs. Transfection of APP^−/− cells with a vector encoding APP restored nuclear import of anMan-containing HS. In WT MEFs and N2a neuroblastoma cells exposed to β- or γ-secretase inhibitors, nuclear translocation was greatly impeded, suggesting involvement of APP/APLP2 degradation products. In Tg2576 MEFs, the β-inhibitor blocked transport, but the γ-inhibitor did not. During chase in ascorbate-free medium, anMan-containing HS disappeared from the nuclei of WT MEFs. Confocal immunofluorescence microscopy showed that they appeared in acidic, LC3-positive vesicles in keeping with an autophagosomal location. There was increased accumulation of anMan-containing HS in nuclei and cytosolic vesicles upon treatment with chloroquine, indicating that HS was degraded in lysosomes. Manipulations of APP expression and processing may have deleterious effects upon HS function in the nucleus.     

RhoG Protein Regulates Glycoprotein VI-Fc Receptor γ-Chain Complex-mediated Platelet Activation and Thrombus Formation

We investigated the mechanism of activation and functional role of a hitherto uncharacterized signaling molecule, RhoG, in platelets. We demonstrate for the first time the expression and activation of RhoG in platelets. Platelet aggregation, integrin αIIbβ3 activation, and α-granule and dense granule secretion in response to the glycoprotein VI (GPVI) agonists collagen-related peptide (CRP) and convulxin were significantly inhibited in RhoG-deficient platelets. In contrast, 2-MeSADP- and AYPGKF-induced platelet aggregation and secretion were minimally affected in RhoG-deficient platelets, indicating that the function of RhoG in platelets is GPVI-specific. CRP-induced phosphorylation of Syk, Akt, and ERK, but not SFK (Src family kinase), was significantly reduced in RhoG-deficient platelets. CRP-induced RhoG activation was consistently abolished by a pan-SFK inhibitor but not by Syk or PI3K inhibitors. Interestingly, unlike CRP, platelet aggregation and Syk phosphorylation induced by fucoidan, a CLEC-2 agonist, were unaffected in RhoG-deficient platelets. Finally, RhoG^−/− mice had a significant delay in time to thrombotic occlusion in cremaster arterioles compared with wild-type littermates, indicating the important in vivo functional role of RhoG in platelets. Our data demonstrate that RhoG is expressed and activated in platelets, plays an important role in GPVI-Fc receptor γ-chain complex-mediated platelet activation, and is critical for thrombus formation in vivo.     

Isolation,cDNA Cloning,and Structure-based Functional Characterization of Oryctin,a Hemolymph Protein from the Coconut Rhinoceros Beetle,Oryctes rhinoceros,as a Novel Serine Protease Inhibitor

We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant ¹³C,¹⁵N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with K_i values of 3.9 × 10⁻¹⁰ m, 6.2 × 10⁻¹⁰ m, 1.4 × 10⁻⁹ m, and 1.2 × 10⁻⁸ m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections.     

Heterotrimeric Kinesin-2 (KIF3) Mediates Transition Zone and Axoneme Formation of Mouse Photoreceptors

Anterograde intraflagellar transport (IFT) employing kinesin-2 molecular motors has been implicated in trafficking of photoreceptor outer segment proteins. We generated embryonic retina-specific (prefix “emb”) and adult tamoxifen-induced (prefix “tam”) deletions of KIF3a and IFT88 in adult mice to study photoreceptor ciliogenesis and protein trafficking. In ^embKif3a^−/− and in ^embIft88^−/− mice, basal bodies failed to extend transition zones (connecting cilia) with outer segments, and visual pigments mistrafficked. In contrast, ^tamKif3a^−/− and ^tamIft88^−/− photoreceptor axonemes disintegrated slowly post-induction, starting distally, but rhodopsin and cone pigments trafficked normally for more than 2 weeks, a time interval during which the outer segment is completely renewed. The results demonstrate that visual pigments transport to the retinal outer segment despite removal of KIF3 and IFT88, and KIF3-mediated anterograde IFT is responsible for photoreceptor transition zone and axoneme formation.     

Crystal Structures of Vertebrate Dihydropyrimidinase and Complexes from Tetraodon nigroviridis with Lysine Carbamylation: METAL AND STRUCTURAL REQUIREMENTS FOR POST-TRANSLATIONAL MODIFICATION AND FUNCTION*

Lysine carbamylation, a post-translational modification, facilitates metal coordination for specific enzymatic activities. We have determined structures of the vertebrate dihydropyrimidinase from Tetraodon nigroviridis (TnDhp) in various states: the apoenzyme as well as two forms of the holoenzyme with one and two metals at the catalytic site. The essential active-site structural requirements have been identified for the possible existence of four metal-mediated stages of lysine carbamylation. Only one metal is sufficient for stabilizing lysine carbamylation; however, the post-translational lysine carbamylation facilitates additional metal coordination for the regulation of specific enzymatic activities through controlling the conformations of two dynamic loops, Ala⁶⁹–Arg⁷⁴ and Met¹⁵⁸–Met¹⁶⁵, located in the tunnel for the substrate entrance. The substrate/product tunnel is in the “open form” in the apo-TnDhp, in the “intermediate state” in the monometal TnDhp, and in the “closed form” in the dimetal TnDhp structure, respectively. Structural comparison also suggests that the C-terminal tail plays a role in the enzymatic function through interactions with the Ala⁶⁹–Arg⁷⁴ dynamic loop. In addition, the structures of the dimetal TnDhp in complexes with hydantoin, N-carbamyl-β-alanine, and N-carbamyl-β-amino isobutyrate as well as apo-TnDhp in complex with a product analog, N-(2-acetamido)-iminodiacetic acid, have been determined. These structural results illustrate how a protein exploits unique lysines and the metal distribution to accomplish lysine carbamylation as well as subsequent enzymatic functions.     

Different pathways of choline metabolism in two choline-independent strains of Streptococcus pneumoniae and their impact on virulence

The two recently characterized Streptococcus pneumoniae strains—R6Chi and R6Cho⁻—that have lost the unique auxotrophic requirement of this bacterial species for choline differ in their mechanisms of choline independence. In strain R6Chi the mechanism is caused by a point mutation in tacF, a gene that is part of the pneumococcal lic2 operon, which is essential for growth and survival of the bacteria. Cultures of lic2 mutants of the encapsulated strain D39Chi growing in choline-containing medium formed long chains, did not autolyze, had no choline in their cell wall, and were completely avirulent in the mouse intraperitoneal model. In contrast, while the Cho⁻ strain carried a complete pneumococcal lic2 operon and had no mutations in the tacF gene, deletion of the entire lic2 operon had no effect on the growth or phenotype of strain Cho⁻. These observations suggest that the biochemical functions normally dependent on determinants of the pneumococcal lic2 operon may also be carried out in strain Cho⁻ by a second set of genetic elements imported from Streptococcus oralis, the choline-independent streptococcal strain that served as the DNA donor in the heterologous transformation event that produced strain R6Cho⁻. The identification in R6Cho⁻ of a large (20-kb) S. oralis DNA insert carrying both tacF and licD genes confirms this prediction and suggests that these heterologous elements may represent a “backup” system capable of catalyzing P-choline incorporation and export of teichoic acid chains under conditions in which the native lic2 operon is not functional.