Myelopoietins (MPOs) are a family of engineered dual interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) receptor agonists that are superior in comparison to the single agonists in their ability to promote the growth and maturation of hematopoietic cells of the myeloid lineage. A series of MPO molecules were created which incorporated circularly permuted G-CSF (cpG-CSF) sequences with an IL-3 receptor (IL-3R) agonist moiety attached at locations that correspond to the loops that connect the helices of the G-CSF four-helix bundle structure. The cpG-CSF linkage sites (using the original sequence numbering) were residue 39, which is at the beginning of the first loop connecting helices 1 and 2; residue 97, which is in the turn connecting helices 2 and 3; and residues 126, 133, and 142, which are at the beginning, middle, and end, respectively, of the loop connecting helices 3 and 4. The N- and C-terminal helices of each cpG-CSF domain were constrained, either by direct linkage of the termini (L0) or by replacement of the amino-terminal 10-residue segment with a seven-residue linker composed of SGGSGGS (L1). All of the MPO molecules stimulated the proliferation of both IL-3-dependent (EC50 = 13-95 pM) and G-CSF-dependent (EC50 = 35-710 pM) cell lines. MPOs with the IL-3R agonist domain linked to cpG-CSFs in the first (residue 39) or second (residue 133) long overhand loops were found by CD spectroscopy to have helical contents similar to that expected for a protein comprised of two linked four-helix bundles. The MPOs retained the ability to bind to the IL-3R with affinities similar to that of the parental MPO. Using both a cell surface competitive binding assay and surface plasmon resonance detection of binding kinetics, the MPOs were found to bind to the G-CSF receptor with low nanomolar affinities, similar to that of G-CSF(S17). In a study of isolated cpG-CSF domains [Feng, Y., et al. (1999) Biochemistry 38, 4553-4563], domains with the L1 linker had lower G-CSF receptor-mediated proliferative activities and conformational stabilities than those which had the L0 linker. A similar trend was found for the MPOs in which the G-CSFR agonist activity is mostly a property of the cpG-CSF domain. Important exceptions were found in which the linkage to the IL-3R agonist domain either restored (e.g., attachment at residue 142) or further decreased (linkage at residue 39) the G-CSFR-mediated proliferative activity. MPO in which the IL-3R agonist domain is attached to the cpG-CSF(L1)[133/132] domain was shown to be more potent than the coaddition of the IL-3R agonist and G-CSF in stimulating the production of CFU-GM colonies in a human bone marrow-derived CD34+ colony-forming unit assay. Several MPOs also had decreased proinflammatory activity in a leukotriene C4 release assay using N-formyl-Met-Leu-Phe-primed human monocytes. It was found that circular permutation of the G-CSF domain can alter the ratio of G-CSFR:IL-3R agonist activities, demonstrating that it is a useful tool in engineering chimeric protein...
In vitro translation products of polyadenylated RNA from untreated and auxin-treated elongating sections of soybean (Glycine max var. Wayne) hypocotyl were analyzed by two-dimensional polyacrylamide gel electrophoresis. The levels of translatable messenger RNA for at least ten in vitro translation products are increased by auxin treatment. The induction by auxin occurs rapidly (within 15 minutes), and the amounts of the induced in vitro translation products increase with time of auxin treatment. Indoleacetic acid has the same effect on the population of translatable messenger RNA as 2,4-dichlorophenoxyacetic acid. The auxin-induced in vitro translation products disappear rapidly when Actinomycin D is present during the last two hours of a three-hour auxin treatment.achieve sufficient radiospecific activity of the polypeptides to allow analysis. Such experimental constraints make it infeasible to study rapid growth effects induced by auxin.In contrast to in vivo radioactive labeling experiments, in vitro translation of purified messenger RNA allows one to assess the spectrum of polypeptides synthesized at discrete and very short times after auxin application. This approach is limited only by how faithfully the in vitro translation system mimics that which is occurring in vivo at any given time. We have thus taken such an approach to determine if auxin modifies the spectrum of polypeptides synthesized during auxin-induced cell extension of soybean hypocotyl. We have utilized both wheat germ and rabbit reticulocyte translation systems and have analyzed the in vitro translation products by two-dimensional polyacrylamide gel electrophoresis.Continued synthesis of RNA and protein is obligatory for auxin-induced cell extension in excised plant organs (i.e. hypocotyls, epicotyls, and coleoptiles) during steady-state growth (I I for review). In addition to this requirement for steady-state growth, protein synthesis is also required for the auxin-induced H+ secretion which presumably elicits the rapid growth response (2, 7, 18). There is, however, little or no concrete information on the types of proteins required for auxin-induced cell extension and on the roles such proteins play in the process of cell extension. Even more basic questions remain unanswered; for example, does auxin simply maintain or enhance general protein synthesis in excised organs, or does auxin alter the types or enhance the synthesis of a limited number ofpolypeptides (ie. "growth essential proteins")? If the synthesis of specific polypeptides required for cell extension is induced by auxin, we might expect these polypeptides to appear rapidly (ie. within 10-15 min after auxin application [8] MATERIALS AND METHODS Plant Material. Soybean seeds (Glycine max var. Wayne) were germinated in a 1:1 mixture of moistened vermiculite and Perlite in the dark at 30°C. After 72 h, the seedlings were harvested, and the 1.2-cm section from the elongating region of the hypocotyls (0.5-1.7 cm below the cotyledons) was excised. The sections were rinsed with...
Two soybean ribulose-1,5-bisphosphate carboxylase small subunit (SSU) genes, SRS1 and SRS4, are highly homologous over a region that includes 4 kb of 5' and 1 kb of 3' flanking sequences. The expression of these genes was compared using synthetic oligonucleotide probes. Analysis of a soybean leaf cDNA library indicates that SRS1 and SRS4 are the most highly expressed members of the soybean SSU gene family. Similar changes were observed in the RNA levels for these genes in response to white light, far-red light and darkness, although SRS1 was expressed at a four-fold higher level in total RNA than SRS4 under all conditions. However, nuclear run-on assays indicate that SRS1 is transcribed at a lower rate than SRS4, which suggests that SRS1 RNA is more stable. S1 nuclease analysis and oligonucleotide directed RNase H cleavage indicate that transcripts from both genes are polyadenylated within two principle regions separated by 35 nt. Sequence analysis of 16 independent cDNA clones identified seven different polyadenylation sites, and six of these sites lie within these two regions. Although SRS1 RNA was poorly recovered during poly(A)+ fractionation, RNase H cleavage experiments showed that transcripts from SRS1 and SRS4 had similar poly (A) tail lengths ranging from 0 to 220 nt. In addition, and despite differences in the untranslated leader sequences, SRS1 and SRS4 RNAs are assembled into polysomes with equal efficiencies. The overall similarity in expression patterns for these two genes further illustrates the coordinate evolution of individual members of a SSU gene family and is consistent with the proposal that gene conversion homogenizes both the coding and regulatory regions of these genes.
The sequence of granulocyte colony-stimulating factor (G-CSF) has been circularly permuted by introducing new chain termini into interhelical loops and by constraining the N- and C-terminal helices, either by direct linkage of the termini (L0) or by substitution of the amino-terminal 10-residue segment with a seven-residue linker composed of glycines and serines (L1). All the circularly permuted G-CSFs (cpG-CSFs) were able to fold into biologically active structures that could recognize the G-CSF receptor. CD and NMR spectroscopy demonstrated that all of the cpG-CSFs adopted a fold similar to that of the native molecule, except for one [cpG-CSF(L1)[142/141]] which has the new termini at the end of loop 34 with the shorter L1 linker. All of the cpG-CSFs underwent cooperative unfolding by urea, and a systematically lower free energy change (DeltaGurea) was observed for molecules with the shorter L1 linker than for those molecules in which the original termini were directly linked (the L0 linker). The thermodynamic stability of the cpG-CSFs toward urea was found to correlate with their relative ability to stimulate proliferation of G-CSF responsive cells. Taken together, these results indicate that the G-CSF sequence is robust in its ability to undergo linear rearrangement and adopt a biologically active conformation. The choice of linker, with its effect on stability, seems to be important for realizing the full biological activity of the three-dimensional structure. The breakpoint and linker together are the ultimate determinants of the structural and biological profiles of these circularly permuted cytokines. In the following paper [McWherter, C. A., et al. (1999) Biochemistry 38, 4564-4571], McWherter and co-workers have used circularly permuted G-CSF sequences to engineer chimeric dual IL-3 and G-CSF receptor agonists in which the relative spatial orientation of the receptor agonist domains is varied. Interpreting the differences in activity for the chimeric molecules in terms of the connectivity between domains depends critically on the results reported here for the isolated cpG-CSF domains.
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