The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

In native chemical ligation, an unprotected peptide α-carboxy thioester is reacted with a second peptide containing an N-terminal cysteine residue. It was anticipated that addition of thiophenol to a native chemical ligation reaction would keep cysteine side chains reduced, catalyze the reversal of unproductive thioester formation, and generate a more reactive phenyl thioester through thiol exchange. Several model peptide−α-thioesters were treated with an excess of a competing thiol to investigate their susceptibility to thiol exchange:  a highly activated 3-nitro-4-carboxybenzyl α-thioester was smoothly converted to the less activated benzyl α-thioester through the addition of an excess of benzyl mercaptan; similarly, a peptide containing the benzyl α-thioester group was converted to a more reactive phenyl α-thioester by addition of thiophenol. Even a weakly activated peptide−α-thioester was converted to a substantially more reactive species, as demonstrated by the conversion of peptide−αCOS-CH2COOH to peptide−αCOS-phenyl. The utility of in situ transthioesterification in native chemical ligation reactions was demonstrated by model syntheses of the 110-residue barnase polypeptide chain. The use of thiophenol as an additive in the ligation gave clean, rapid reaction to form the desired amide-linked product in high yield. The in situ transthioesterification process is broadly applicable to the total chemical synthesis of proteins by native chemical ligation.

Understanding the pathogenesis of cancer-related bone disease is crucial to the discovery of new therapies. Here we identify activin A, a TGF-β family member, as a therapeutically amenable target exploited by multiple myeloma (MM) to alter its microenvironmental niche favoring osteolysis. Increased bone marrow plasma activin A levels were found in MM patients with osteolytic disease. MM cell engagement of marrow stromal cells enhanced activin A secretion via adhesion-mediated JNK activation. Activin A, in turn, inhibited osteoblast differentiation via SMAD2-dependent distalless homeobox-5 down-regulation. Targeting activin A by a soluble decoy receptor reversed osteoblast inhibition, ameliorated MM bone disease, and inhibited tumor growth in an in vivo humanized MM model, setting the stage for testing in human clinical trials.osteoblasts | osteoclasts | tumor niche

In the oxidized "ES" state of cytochrome c peroxidase, Trp-191 is reversibly oxidized to a stable cation free radical by the hypervalent heme. To explore the potential for engineering a binding site for heterocyclic compounds at this site, the mutant W191G was constructed. Two independent crystal structures of W191G at 2.1- and 2.3-A resolution show that W191G contains a well-defined, approximately 180-A3 cavity at the Trp-191 site. The cavity is occupied by five ordered water molecules which participate in an extensive hydrogen-bonding network with each other, with polar main-chain atoms, and with the carboxylate of Asp-235. After a number of heterocyclic compounds were screened, evidence was obtained that substituted imidazoles bind to the cavity of W191G. Titration of W191G with imidazole resulted in a perturbation of the Soret absorption band that was not observed for W191H, W191F, or the native enzyme. The dissociation constants for binding of benzimidazole, imidazole, 2-ethylimidazole, 1-methylimidazole, 2-methylimidazole, and 1,2-dimethylimidazole to W191G were respectively 2.58, 0.70, 0.36, 0.057, 0.047, and 0.027 mM at pH 6.0. The highest binding affinity was exhibited by 1,2-dimethylimidazole, indicating that steric interactions and the efficiency of filling the cavity are important determinants for specificity. The Kd for imidazole binding increased from 0.7 mM at pH 6 to 3.0 mM at pH 8 and could be fit to a single proton ionization curve with a pKa of 7.4, demonstrating the preferential binding by the imidazolium ion (pKa = 7.3). The binding of a number of substituted imidazoles to the cavity of W191G was verified by X-ray crystallographic analysis. The most clearly defined density was observed for W191G crystals soaked in 1 mM 1,2-dimethylimidazole and was consistent with an oriented occupation in which the unsubstituted nitrogen forms a hydrogen bond or ion pair interaction with Asp-235. Thus, enhanced binding of positively charged molecules may be the result of interactions with this carboxylate. An analogous interaction may stabilize the developing positive charge on the Trp-191 radical of the wild-type enzyme. While the oxidation of imidazoles by the ferryl intermediate of W191G was neither expected nor observed, this study has defined the structural determinants for small molecule binding to an artificially created cavity near a heme center which is capable of generating oxidized species at a potential of over 1 V, and these results will guide future attempts for novel substrate oxidation by CCP.