HindIII Restriction Site Research Articles

Structure of the Bacteriophage φKZ Lytic Transglycosylase gp144

Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage phi KZ has been determined to 2.5-A resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. phi KZ gp144 is a 260-residue alpha-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G D-Ala-D-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu 115 in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the phi KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine)(4) has been determined to 2.6-A resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.

The Key Residue for Substrate Transport (Glu14) in the EmrE Dimer Is Asymmetric

Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli multidrug transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms a functional dimer to transport a diverse array of aromatic, positively charged substrates in a proton/drug antiport fashion. Here, we report (13)C chemical shifts of the essential residue Glu(14) within the binding pocket. To ensure a native environment, EmrE was reconstituted into E. coli lipids. Experiments were carried out using one- and two-dimensional double quantum filtered (13)C solid state NMR. For an unambiguous assignment of Glu(14), an E25A mutation was introduced to create a single glutamate mutant. Glu(14) was (13)C-labeled using cell-free expression. Purity, labeling, homogeneity, and functionality were probed by mass spectrometry, NMR spectroscopy, freeze fracture electron microscopy, and transport assays. For Glu(14), two distinct sets of chemical shifts were observed that indicates structural asymmetry in the binding pocket of homodimeric EmrE. Upon addition of ethidium bromide, chemical shift changes and altered line shapes were observed, demonstrating substrate coordination by both Glu(14) in the dimer.

Amino Acids Allosterically Regulate the Thiamine Diphosphate-dependent α-Keto Acid Decarboxylase from Mycobacterium tuberculosis

The gene rv0853c from Mycobacterium tuberculosis strain H37Rv codes for a thiamine diphosphate-dependent alpha-keto acid decarboxylase (MtKDC), an enzyme involved in the amino acid degradation via the Ehrlich pathway. Steady state kinetic experiments were performed to determine the substrate specificity of MtKDC. The mycobacterial enzyme was found to convert a broad spectrum of branched-chain and aromatic alpha-keto acids. Stopped-flow kinetics showed that MtKDC is allosterically activated by alpha-keto acids. Even more, we demonstrate that also amino acids are potent activators of this thiamine diphosphate-dependent enzyme. Thus, metabolic flow through the Ehrlich pathway can be directly regulated at the decarboxylation step. The influence of amino acids on MtKDC catalysis was investigated, and implications for other thiamine diphosphate-dependent enzymes are discussed.

The Structure of a Full-length Response Regulator from Mycobacterium tuberculosis in a Stabilized Three-dimensional Domain-swapped, Activated State

The full-length, two-domain response regulator RegX3 from Mycobacterium tuberculosis is a dimer stabilized by three-dimensional domain swapping. Dimerization is known to occur in the OmpR/PhoB subfamily of response regulators upon activation but has previously only been structurally characterized for isolated receiver domains. The RegX3 dimer has a bipartite intermolecular interface, which buries 2357 A(2) per monomer. The two parts of the interface are between the two receiver domains (dimerization interface) and between a composite receiver domain and the effector domain of the second molecule (interdomain interface). The structure provides support for the importance of threonine and tyrosine residues in the signal transduction mechanism. These residues occur in an active-like conformation stabilized by lanthanum ions. In solution, RegX3 exists as both a monomer and a dimer in a concentration-dependent equilibrium. The dimer in solution differs from the active form observed in the crystal, resembling instead the model of the inactive full-length response regulator PhoB.

CD4 Interacts Constitutively with Multiple CCR5 at the Plasma Membrane of Living Cells

The entry of human immunodeficiency virus into target cells requires successive interactions of the viral envelope glycoprotein gp120 with CD4 and the chemokine receptors CCR5 or CXCR4. We previously demonstrated, by Förster resonance energy transfer experiments, the constitutive association of CD4 and CCR5 at the surface of living cells. We therefore speculated that this interaction may correlate with compartmentalization of CD4 and CCR5 within the plasma membrane. Here, we characterize the lateral distribution, the dynamics, and the stoichiometry of these receptors in living cells stably expressing CD4 and/or CCR5 by means of fluorescence recovery after photobleaching at variable radii experiments. We found that (i) these receptors expressed alone are confined into 1-microm-sized domains, (ii) CD4-CCR5 associations occur outside and inside smaller domains, and (iii) these interactions involve multiple CCR5 molecules per CD4.

Dynamic Receptor-dependent Activation of Inducible Nitric-oxide Synthase by ERK-mediated Phosphorylation of Ser745

Nitric oxide (NO) is a pleiotropic regulator of vascular function, and its overproduction by inducible nitric-oxide synthase (iNOS) in inflammatory conditions plays an important role in the pathogenesis of vascular diseases. iNOS activity is thought to be regulated primarily at the level of expression to generate "high output" NO compared with constitutive NO synthases. Here we show iNOS activity is acutely up-regulated by activation of the B1-kinin receptor (B1R) in human endothelial cells or transfected HEK293 cells to generate 2.5-5-fold higher NO than that stimulated by Arg alone. Increased iNOS activity was dependent on B1R activation of the MAPK ERK. In HEK293 cells transfected with human iNOS and B1R, ERK phosphorylated iNOS on Ser745 as determined by Western analysis using phospho-Ser antibody, in vitro kinase assays with activated ERK, and MALDI-TOF mass spectrometry. Mutation of Ser745 to Ala did not affect basal iNOS activity but eliminated iNOS phosphorylation and activation in response to B1R agonist. Mutation of Ser745 to Asp resulted in a basally hyperactive iNOS whose activity was not further increased by B1R agonist. ERK and phospho-ERK (after B1R activation) were co-localized with iNOS as determined by confocal fluorescence microscopy. Furthermore, ERK co-immunoprecipitated with iNOS. The discovery that iNOS can be phosphorylated by ERK and acutely activated by receptor-mediated signaling reveals a new level of regulation for this isoform. These findings provide a novel therapeutic target to explore in the treatment of vascular inflammatory diseases.

Knockdown of the Intraflagellar Transport Protein IFT46 Stimulates Selective Gene Expression in Mouse Chondrocytes and Affects Early Development in Zebrafish

Bone morphogenetic proteins (BMPs) act as multifunctional regulators in morphogenesis during development. In particular they play a determinant role in the formation of cartilage molds and their replacement by bone during endochondral ossification. In cell culture, BMP-2 favors chondrogenic expression and promotes hypertrophic maturation of chondrocytes. In mouse chondrocytes we have identified a BMP-2-sensitive gene encoding a protein of 301 amino acids. This protein, named mIFT46, is the mouse ortholog of recently identified Caenorhabditis elegans and Chlamydomonas reinhardtii intraflagellar transport (IFT) proteins. After generation of a polyclonal antibody against mIFT46, we showed for the first time that the endogenous protein is located in the primary cilium of chondrocytes. We also found that mIFT46 is preferentially expressed in early hypertrophic chondrocytes located in the growth plate. Additionally, mIFT46 knockdown by small interfering RNA oligonucleotides in cultured chondrocytes specifically stimulated the expression of several genes related to skeletogenesis. Furthermore, Northern blotting analysis indicated that mIFT46 is also expressed before chondrogenesis in embryonic mouse development, suggesting that the role of mIFT46 might not be restricted to cartilage. To explore the role of IFT46 during early development, we injected antisense morpholino oligonucleotides in Danio rerio embryos to reduce zebrafish IFT46 protein (zIFT46) synthesis. Dramatic defects in embryonic development such as a dorsalization and a tail duplication were observed. Thus our results taken together indicate that the ciliary protein IFT46 has a specific function in chondrocytes and is also essential for normal development of vertebrates.

Membrane-type 1-Matrix Metalloproteinase Regulates Intracellular Adhesion Molecule-1 (ICAM-1)-mediated Monocyte Transmigration

We examined the mechanism regulating intercellular cell adhesion molecule-1 (ICAM-1)-dependent monocyte transendothelial migration. Monocyte migration through endothelial cells expressing ICAM-1 alone was comparable to that of tumor necrosis factor-alpha-treated cells. Transmigration was reduced in ICAM-1 lacking the cytoplasmic tail and in tyrosine to alanine substitutions at Tyr-485 and Tyr-474. Tissue inhibitors of matrix metalloproteinases (TIMPs) -2 and -3 blocked transmigration, whereas TIMP-1 was ineffective. This profile suggested a role for membrane-type matrix metalloproteinases (MT-MMPs) in transmigration. Inhibitory antibodies and small interference RNA directed against MT1-MMP blocked transmigration, whereas overexpression of MT1-MMP in endothelial cells or monocytes promoted transmigration. MT1-MMP mediated the ectodomain cleavage of ICAM-1 that was blocked by TIMP-2 and -3. Overexpression of MT1-MMP rescued function in ICAM-1Y485A, and to a lesser extent in the cytoplasmic tail-deleted ICAM-1. In a binding assay, wild-type ICAM-1 bound to purified MT1-MMP while ICAM-1 mutants bound poorly. MT1-MMP co-localized with ICAM-1 at distinct structures in endothelial cells. MT1-MMP localization with cells expressing ICAM-1 mutations was reduced and diffused. These results indicate that the cytoplasmic tail of ICAM-1 regulates leukocyte transmigration through MT1-MMP interaction.

Discovery and Characterization of a Second Mammalian Thiol Dioxygenase, Cysteamine Dioxygenase

There are only two known thiol dioxygenase activities in mammals, and they are ascribed to the enzymes cysteine dioxygenase (CDO) and cysteamine (2-aminoethanethiol) dioxygenase (ADO). Although many studies have been dedicated to CDO, resulting in the identification of its gene and even characterization of the tertiary structure of the protein, relatively little is known about cysteamine dioxygenase. The failure to identify the gene for this protein has significantly hampered our understanding of the metabolism of cysteamine, a product of the constitutive degradation of coenzyme A, and the synthesis of taurine, the final product of cysteamine oxidation and the second most abundant amino acid in mammalian tissues. In this study we identified a hypothetical murine protein homolog of CDO (hereafter called ADO) that is encoded by the gene Gm237 and belongs to the DUF1637 protein family. When expressed as a recombinant protein, ADO exhibited significant cysteamine dioxygenase activity in vitro. The reaction was highly specific for cysteamine; cysteine was not oxidized by the enzyme, and structurally related compounds were not competitive inhibitors of the reaction. When overexpressed in HepG2/C3A cells, ADO increased the production of hypotaurine from cysteamine. Similarly, when endogenous expression of the human ADO ortholog C10orf22 in HepG2/C3A cells was reduced by RNA-mediated interference, hypotaurine production decreased. Western blots of murine tissues with an antibody developed against ADO showed that the protein is ubiquitously expressed with the highest levels in brain, heart, and skeletal muscle. Overall, these data suggest that ADO is responsible for endogenous cysteamine dioxygenase activity.

The Mast Cell-restricted Tryptase mMCP-6 Has a Critical Immunoprotective Role in Bacterial Infections

Although it has been shown that mast cell-deficient mice have diminished innate immune responses against bacteria, the most important immunoprotective factors secreted from activated mast cells have not been identified. Mouse mast cell protease 6 is a tetramer-forming tryptase. This serine protease is abundant in the secretory granules and is exocytosed upon bacterial challenge. Here we have described the generation of a mast cell protease-6-null mouse. Our discovery that mice lacking this neutral protease cannot efficiently clear Klebsiella pneumoniae from their peritoneal cavities reveals an essential role for this serine protease, and presumably its human ortholog, in innate immunity.

E3-Independent Monoubiquitination of Ubiquitin-Binding Proteins

Ubiquitin (Ub)-binding domains (UBDs) are key elements in conveying Ub-based cellular signals. UBD-containing proteins interact with ubiquitinated targets and control numerous biological processes. They themselves undergo UBD-dependent monoubiquitination, which promotes intramolecular binding of the UBD to the attached Ub and leads to their inactivation. Here, we report that, in contrast to the established ubiquitination pathway, the presence of UBDs allows the ubiquitination of host proteins independently of E3 ligases. UBDs of different types, including UBA, UIM, UBM, NFZ, and UBZ, can directly cooperate with Ub-charged E2 enzymes to promote monoubiquitination. Using FRET and siRNA technologies, we verify that Ub-loaded E2 and substrates interact in cells and that E2 enzymes are essential for their monoubiquitination in vivo. This modification is mechanistically and functionally distinct from E3-mediated and growth factor-dependent monoubiquitination.

An Essential Cell Cycle-regulated Nucleolar Protein Relocates to the Mitotic Spindle Where It Is Involved in Mitotic Progression in Trypanosoma brucei

TbNOP86 and TbNOP66 are two novel nucleolar proteins isolated in Trypanosoma brucei. They share 92.6% identity, except for an additional C-terminal domain of TbNOP86 of 182 amino acids in length. Both proteins are found in Trypanosomatidae, but similarity to other eukaryotic proteins could not be found. TbNOP86 and TbNOP66 are expressed at similar level in procyclic and bloodstream forms, although the relative level of expression of TbNOP66 is 11 times lower. TbNOP86 undergoes post-translational modifications, as it is found predominantly at 110 kDa compared with the predicted 86 kDa. Immunofluorescence of overexpressed ty-tagged TbNOP86 and TbNOP66 showed that both proteins accumulated in the nucleolus of G(1) cells. This was confirmed by the co-localization of an endogenous TbNOP86-myc with the nucleolar protein Nopp140. TbNOP86-ty localization is cell cycle-regulated, because it colocalizes with the mitotic spindle in mitotic cells. TbNOP86 is required for mitotic progression in both life stages as depleted cells are enriched in the G(2)/M phase. In procyclic cells, a reduced growth rate is accompanied by an accumulation of zoids (0N1K), 2N1K, and multinucleated cells (xNyK). The 2N1K cells are blocked in late mitosis as nucleolar segregation is completed. TbNOP86 depletion in bloodstream form caused a drastic growth inhibition producing cells bearing two kinetoplasts and an enlarged nucleus (1N(*)2K), followed by an accumulation of 2N2K cells with connected nuclei and xNyK cells. These studies of TbNOP86 provide a more comprehensive account of proteins involved in mitotic events in trypanosomes and should lead to the identification of partners with similar function.

Actin-binding Protein α-Actinin-1 Interacts with the Metabotropic Glutamate Receptor Type 5b and Modulates the Cell Surface Expression and Function of the Receptor

Receptors for neurotransmitters require scaffolding proteins for membrane microdomain targeting and for regulating receptor function. Using a yeast two-hybrid screen, alpha-actinin-1, a major F-actin cross-linking protein, was identified as a binding partner for the C-terminal domain of metabotropic glutamate receptor type 5b (mGlu(5b) receptor). Co-expression, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between mGlu(5b) receptor and alpha-actinin-1 in both transfected HEK-293 cells and rat striatum. The interaction of alpha-actinin-1 with mGlu(5b) receptor modulated the cell surface expression of the receptor. This was dependent on the binding of alpha-actinin-1 to the actin cytoskeleton. In addition, the alpha-actinin-1/mGlu(5b) receptor interaction regulated receptor-mediated activation of the mitogen-activated protein kinase pathway. Together, these findings indicate that there is an alpha-actinin-1-dependent mGlu(5b) receptor association with the actin cytoskeleton modulating receptor cell surface expression and functioning.

Heat Shock Factor 2 (HSF2) Contributes to Inducible Expression of hsp Genes through Interplay with HSF1

The heat shock response is a defense reaction activated by proteotoxic damage induced by physiological or environmental stress. Cells respond to the proteotoxic damage by elevated expression of heat shock proteins (Hsps) that function as molecular chaperones and maintain the vital homeostasis of protein folds. Heat shock factors (HSFs) are the main transcriptional regulators of the stress-induced expression of hsp genes. Mammalian HSF1 was originally identified as the transcriptional regulator of the heat shock response, whereas HSF2 has not been implicated a role in the stress response. Previously, we and others have demonstrated that HSF1 and HSF2 interact through their trimerization domains, but the functional consequence of this interaction remained unclear. We have now demonstrated on chromatin that both HSF1 and HSF2 were able to bind the hsp70 promoter not only in response to heat shock but also during hemin-induced differentiation of K562 erythroleukemia cells. In both cases an intact HSF1 was required in order to reach maximal levels of promoter occupancy, suggesting that HSF1 influences the DNA binding activity of HSF2. The functional consequence of the HSF1-HSF2 interplay was demonstrated by real-time reverse transcription-PCR analyses, which showed that HSF2 was able to modulate the HSF1-mediated expression of major hsp genes. Our results reveal, contrary to the predominant model, that HSF2 indeed participates in the transcriptional regulation of the heat shock response.

The Influence of Insertion of a Critical Residue (Arg356) in Structure and Bioluminescence Spectra of Firefly Luciferase

The firefly bioluminescence reaction, which uses luciferin, Mg-ATP, and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by luciferase and visible light is emitted. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Among different beetle luciferases, those from Phrixothrix railroad worm emit either yellow or red bioluminescence colors. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional Arg residue at 353, which is absent in firefly luciferases. We report here the construction and purification of a mutant at residue Arg(356), which is not conserved in beetle luciferases. By insertion of an additional residue (Arg(356)) using site-specific insertion mutagenesis in a green-emitter luciferase (Lampyris turkestanicus) the color of emitted light was changed to red and the optimum temperature of activity was also increased. Insertion of this Arg in an important flexible loop showed changes of the bioluminescence color and the luciferase reaction took place with relatively retention of its basic kinetic properties such as Km and relative activity. Comparison of native and mutant luciferases using homology modeling reveals a significant conformational change of the flexible loop in the red mutant. Movement of flexible loop brought about a new ionic interaction concomitant with a change in polarity of the emitter site, thereby leading to red emission. It is worthwhile to note that the increased optimum temperature and emission of red light might make mutant luciferase a suitable reporter for the study of gene expression and bioluminescence imaging.

Reduction of Carboxylic Acids by Nocardia Aldehyde Oxidoreductase Requires a Phosphopantetheinylated Enzyme

Aldehyde oxidoreductase (carboxylic acid reductase (Car)) catalyzes the magnesium-, ATP-, and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes. Heterologous expression of the car gene in Escherichia coli afforded purified recombinant enzyme with a specific activity nearly 50-fold lower than that of purified native Nocardia sp. enzyme. The 5-fold increase in specific activity obtained by incubating purified recombinant Car with CoA and Nocardia cell-free extracts indicated that post-translational phosphopantetheinylation of Car is required for maximum enzyme activity. Nocardia phosphopantetheine transferase (PPTase) expressed in E. coli was isolated and characterized. When incubated with [(3)H]acetyl-CoA and Nocardia PPTase, the labeled acetylphosphopantetheine moiety was incorporated into recombinant Car. Coexpression of Nocardia Car and PPTase in E. coli gave a reductase with nearly 20-fold higher specific activity. Site-directed mutagenesis in which Ser(689) was replaced with Ala resulted in an inactive Car mutant. The results show that Car expressed in Escherichia coli is an apoenzyme that is converted to a holoenzyme by post-translational modification via phosphopantetheinylation. Doubly recombinant resting E. coli cells efficiently reduce vanillic acid to vanillin.

Studies of an Association in Boys of Blood Pressure and the Y Chromosome

Variations in the Y chromosome have shown significant, albeit inconsistent, associations with hypertension in men. Studies of peripubertal subjects might reveal more information on the origins of the Y chromosome's influence on blood pressure (BP). In the current study, we examined the association of a HindIII restriction site on the Y chromosome with BP in a cohort of young men. Pubertal growth-based analyses were performed in 80 unrelated males with multiple measurements spanning time points beginning before and extending to after their pubertal growth. Blood pressure, height, and weight were measured approximately every 6 months. The period of pubertal growth was used to approximate the time for puberty. The association of the HindIII restriction site to systolic and diastolic BP was determined using a mixed model ANOVA. Estimated systolic and diastolic BPs were higher in HindIII(-) males in the period before, as well as in the period after pubertal growth (systolic BP 2.6 +/- 1.3 mm Hg higher, P < .05), and diastolic BP 2.3 +/- 1.2 mm Hg higher, P = .050). In addition, HindIII(-) males were younger at onset of peak height velocity (12.7 +/- 0.1 v 13.3 +/- 0.1 years, P < .001). In African Americans, an earlier age of maximal height velocity was associated with a higher systolic and diastolic BP (P < .05 and .02, respectively). The results suggest that variations in genes on the Y chromosome may affect BP in peripubertal males, possibly even before onset of puberty.

Association of Kv1.5 and Kv1.3 Contributes to the Major Voltage-dependent K+ Channel in Macrophages

Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses.

G Protein-coupled Receptor (GPCR) Kinase 2 Regulates Agonist-independent Gq/11 Signaling from the Mouse Cytomegalovirus GPCR M33

The mouse cytomegalovirus M33 protein is highly homologous to mammalian G protein-coupled receptors (GPCRs) yet functions in an agonist-independent manner to activate a number of classical GPCR signal transduction pathways. M33 is functionally similar to the human cytomegalovirus-encoded US28 GPCR in its ability to induce inositol phosphate accumulation, activate NF-kappaB, and promote smooth muscle cell migration. This ability to promote cellular migration suggests a role for viral GPCRs like M33 in viral dissemination in vivo, and accordingly, M33 is required for efficient murine cytomegalovirus replication in the mouse. Although previous studies have identified several M33-induced signaling pathways, little is known regarding the membrane-proximal events involved in signaling and regulation of this receptor. In this study, we used recombinant retroviruses to express M33 in wild-type and Galpha(q/11)(-/-) mouse embryonic fibroblasts and show that M33 couples directly to the G(q/11) signaling pathway to induce high levels of total inositol phosphates in an agonist-independent manner. Our data also show that GRK2 is a potent regulator of M33-induced G(q/11) signaling through its ability to phosphorylate M33 and sequester Galpha(q/11) proteins. Taken together, the results from this study provide the first genetic evidence of a viral GPCR coupling to a specific G protein signaling pathway as well as identify the first viral GPCR to be regulated specifically by both the catalytic activity of the GRK2 kinase domain and the Galpha(q/11) binding activity of the GRK2 RH domain.

PEGylation and Multimerization of the Anti-p185HER-2 Single Chain Fv Fragment 4D5

A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.