Transforming Growth Factor – beta (TGFβ) superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs), and Bone Morphogenetic Proteins (BMPs), are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer), to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values.

Proteins that are released from cells consist of those in the extracellular matrix, as well as extracellular signaling and adhesion molecules. The majority of these extracellular proteins are, however, unknown. To determine their identity, we have used a proteomics approach to define proteins released from neurons, astrocytes and neural precursor cells. Using two-dimensional gels and liquid chromatography/mass spectrometry technology, it is shown that while astrocytes release a relatively small number of proteins, neurons and neuronal precursor cells release a larger number of proteins with more functional diversity. Although there is overlap between the different cell types, the exact composition of the extracellular protein pool is unique for each cell population. The various subsets of extracellular neural proteins include those involved in cellular Redox regulation and chaperones. In addition, many proteolytic enzymes are found outside of the cell. These data show that the extracellular space within the nervous system has a more diverse protein composition than previously thought. Keywords: astrocytes, neuronal cells, neurons, proteomics, secreted proteins. proteins and their diversity was much greater with the neural cells than with mesodermal cells such as muscle and fibroblasts. In addition, unlike nerve cells, the secreted proteins were very similar between the different mesodermal cell types. It was concluded that much of the protein diversity in the nervous system is in proteins that are found in the extracellular space. However, at the time of that publication, the technology was not available to readily determine the identity of this extracellular protein population. Indeed, no studies have previously identified and compared secreted proteins from CNS cell populations. We have therefore used 2-D gel and liquid chromatography/mass spectrometry (LC/ MS) technology to identify a subset of extracellular proteins released by cortical nerve and astrocytes as well as by several clonal nerve precursor cell populations isolated from the embryonic and adult rat cortex. Of the 200 proteins identified, it is shown that a large number of proteins with molecular chaperone and antioxidant properties are secreted, and that distinct subsets of proteins are secreted by nerve, astrocytes, and neural precursor cells. Materials and methods Cell LinesCell lines and preparation of secreted proteins The nerve-like precursor cells B35, B50, B103, and B104 were cloned from nitrosoethylurea-induced brain tumors (Schubert 1974). The major criterion on which the classification of the nerve cells was made was electrical excitability and veratridine-stimulated sodium flux. A large number of other markers, such as cell-surface antigens, are in agreement with the physiology (Schubert et al. 1986). L6 myoblasts were derived from neonatal skeletal muscle (Yaffe 1968).The clonal adult rat hippocampal cell line, adult hippocampal precursor (AHP), was obtained from J. Ray (Salk Institute) (Ray et al. 1993;Taupin et al. 2000). Cel...

Activins and bone morphogenetic proteins (BMPs) are members of the transforming growth factor-␤ family of growth and differentiation factors that induce signaling in target cells by assembling type II and type I receptors at the cell surface. Ligand residues involved in type II binding are located predominantly in the C-terminal region that forms an extended ␤-sheet, whereas residues involved in type I binding are located in the ␣-helical and preceding loop central portion of the molecule. To test whether the central residues are sufficient to determine specificity toward type I receptors, activin A/BMP chimeras were constructed in which the central residues (45-79) of activin A were replaced with corresponding residues of BMP2 and BMP7. The chimeras were assessed for activin type II receptor (Act RII) binding, activin-like bioactivity, and BMP-like activity as well as antagonistic properties toward activin A and myostatin. ActA/BMP7 chimera retained Act RII binding affinity comparable with wild type activin A, whereas ActA/BMP2 chimera showed a slightly reduced affinity toward Act RII. Both the chimeras were devoid of significant activin bioactivity in 293T cells in the A3 Lux reporter assay up to concentrations 10-fold higher than the minimal effective activin A concentration (ϳ4 nM). In contrast, these chimeras showed BMP-like activity in a BRE-Luc assay in HepG2 cells as well as induced osteoblast-like phenotype in C2C12 cells expressing alkaline phosphatase. Furthermore, both the chimeras activated Smad1 but not Smad2 in C2C12 cells. Also, both the chimeras antagonized ligands that signal via activin type II receptor, such as activin A and myostatin. These data indicate that activin residues in the central region determine its specificity toward type I receptors. ActA/BMP chimeras can be useful in the study of receptor specificities and modulation of transforming growth factor-␤ members, activins, and BMPs.

Activins are pluripotent hormones/growth factors that belong to the TGF-β superfamily of growth and differentiation factors. They play a role in cell growth, differentiation and apoptosis, endocrine function, metabolism, wound repair, immune responses, homeostasis, mesoderm induction, bone growth and many other biological processes. Activins and the related bone morphogenic proteins (BMPs) transduce their signal through two classes of single transmembrane receptors. The receptors possess intracellular serine/threonine kinase domains. Signaling occurs when the constitutively active type II kinase domain phosphorylates the type I receptor, which upon activation phosphorylates intracellular signaling molecules. To generate antagonistic ligands, we generated chimeric molecules that disrupt the receptor interactions and thereby the phosphorylation events. The chimeras were designed based on available structural data to maintain high affinity binding to type II receptors. The predicted type I receptor interaction region was replaced by residues present in inactive homologs or in related ligands with different type I receptor affinities.