Signaling by a non-dissociated complex of G protein βγ and α subunits stimulated by a receptor-independent activator of G protein signaling, AGS8

Accumulating evidence suggests that heterotrimeric G protein activation may not require G protein subunit dissociation. Results presented here provide evidence for a subunit dissociation-independent mechanism for G protein activation by a receptor-independent activator of G protein signaling, AGS8. AGS8 is a member of the AGS group III family of AGS proteins thought to activate G protein signaling primarily through interactions with Gbetagamma subunits. Results are presented demonstrating that AGS8 binds to the effector and alpha subunit binding "hot spot" on Gbetagamma yet does not interfere with Galpha subunit binding to Gbetagamma or phospholipase C beta2 activation. AGS8 stimulates activation of phospholipase C beta2 by heterotrimeric Galphabetagamma and forms a quaternary complex with Galpha(i1), Gbeta(1)gamma(2), and phospholipase C beta2. AGS8 rescued phospholipase C beta binding and regulation by an inactive beta subunit with a mutation in the hot spot (beta(1)(W99A)gamma(2)) that normally prevents binding and activation of phospholipase C beta2. This demonstrates that, in the presence of AGS8, the hot spot is not used for Gbetagamma interactions with phospholipase C beta2. Mutation of an alternate binding site for phospholipase C beta2 in the amino-terminal coiled-coil region of Gbetagamma prevented AGS8-dependent phospholipase C binding and activation. These data implicate a mechanism for AGS8, and potentially other Gbetagamma binding proteins, for directing Gbetagamma signaling through alternative effector activation sites on Gbetagamma in the absence of subunit dissociation.     

Construction and characterization of a fusion protein of single-chain anti-carcinoma antibody 323/A3 and human β-glucuronidase

 We report the construction and expression of a fusion protein between a single-chain antibody specific for human carcinomas and human β-glucuronidase by recombinant DNA technology. The sequences encoding the murine monoclonal antibody 323/A3 light- and heavy-chain variable genes were joined by a synthetic sequence encoding a 15-amino-acid linker and combined with human β-glucuronidase by a synthetic sequence encoding a 6-amino-acid linker. The construct was placed under the control of the cytomegalovirus promotor and expressed in COS-7 cells. The yield of active fusion protein was 10 ng/ml transfectoma supernatant. Antibody affinity, antibody specificity and enzyme activity were fully retained by the fusion protein. Biochemical characterization of the fusion protein by sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed a molecular mass of 100 kDa under denaturing conditions. Gel-filtration analysis indicated that the enzymatically active form is a tetramer of approximately 400 kDa. The non-toxic prodrug N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-β-glucuronyl carbamate was activated to the cytotoxic drug doxorubicin by the fusion protein with a hydrolysis rate similar to that of human β-glucuronidase. The growth inhibition of tumor cells coated with the fusion protein and exposed to prodrug was similar to that obtained with doxorubicin. This study shows the feasibility of constructing eukaryotic fusion proteins consisting of a single-chain antibody and human β-glucuronidase for use in the specific activation of anticancer prodrugs. Received: 5 June 1997 / Accepted: 25 October 1997     

Tomato SlSnRK1 protein interacts with and phosphorylates βC1, a pathogenesis protein encoded by a geminivirus β-satellite

The βC1 protein of tomato yellow leaf curl China β-satellite functions as a pathogenicity determinant. To better understand the molecular basis of βC1 in pathogenicity, a yeast two-hybrid screen of a tomato (Solanum lycopersicum) cDNA library was carried out using βC1 as bait. βC1 interacted with a tomato SUCROSE-NONFERMENTING1-related kinase designated as SlSnRK1. Their interaction was confirmed using a bimolecular fluorescence complementation assay in Nicotiana benthamiana cells. Plants overexpressing SnRK1 were delayed for symptom appearance and contained lower levels of viral and satellite DNA, while plants silenced for SnRK1 expression developed symptoms earlier and accumulated higher levels of viral DNA. In vitro kinase assays showed that βC1 is phosphorylated by SlSnRK1 mainly on serine at position 33 and threonine at position 78. Plants infected with βC1 mutants containing phosphorylation-mimic aspartate residues in place of serine-33 and/or threonine-78 displayed delayed and attenuated symptoms and accumulated lower levels of viral DNA, while plants infected with phosphorylation-negative alanine mutants contained higher levels of viral DNA. These results suggested that the SlSnRK1 protein attenuates geminivirus infection by interacting with and phosphorylating the βC1 protein.     

Cell-free production and characterisation of human uncoupling protein 1–3

The uncoupling proteins (UCPs) leak protons across the inner mitochondrial membrane, thus uncoupling the proton gradient from ATP synthesis. The main known physiological role for this is heat generation by UCP1 in brown adipose tissue. However, UCPs are also believed to be important for protection against reactive oxygen species, fine-tuning of metabolism and have been suggested to be involved in disease states such as obesity, diabetes and cancer.Structural studies of UCPs have long been hampered by difficulties in sample preparation with neither expression in yeast nor refolding from inclusion bodies in E. coli yielding sufficient amounts of pure and stable protein. In this study, we have developed a protocol for cell-free expression of human UCP1, 2 and 3, resulting in 1 mg pure protein per 20 mL of expression media. Lauric acid, a natural UCP ligand, significantly improved protein thermal stability and was therefore added during purification. Secondary structure characterisation using circular dichroism spectroscopy revealed the proteins to consist of mostly α-helices, as expected. All three UCPs were able to bind GDP, a well-known physiological inhibitor, as shown by the Fluorescence Resonance Energy Transfer (FRET) technique, suggesting that the proteins are in a natively folded state.     

Do myc,fos and E1A function as protein phosphatase inhibitors?

The oncogenic proteins myc, fos and E1A bear striking resemblance to protein phosphatase inhibitors 1 and 2. Both sets of proteins possess several regions rich in proline (P), glutamic acid (E), serine (S) and threonine (T). In addition to PEST sequences four of the five proteins contain clusters of arginine-arginine pairs. On the basis of these similarities, I suggest that myc, fos and E1A are protein phosphatase inhibitors.