Rapid Cryopurification of the Yeast Mitochondrial Ribosome.

Cryogenic milling, or cryomilling, involves the use of liquid nitrogen to lower the temperature of the biological material and/or the milling process. When applied to the study of subcellular or suborganellar structures and processes, it allows for their rapid extraction from whole cells frozen in the physiological state of choice. This approach has proven to be useful for the study of yeast mitochondrial ribosomes. Following cryomilling of 100 mL of yeast culture, conveniently tagged mitochondrial ribosomes can be immunoprecipitated and purified in native conditions. These ribosomes are suitable for the application of downstream approaches. These include mitoribosome profiling to analyze the mitochondrial translatome or mass spectrometry analyses to assess the mitoribosome proteome in normal growth conditions or under stress, as described in this method.

Novel high molecular weight albumin-conjugated angiotensin II activates ß-arrestin and G-protein pathways.

PURPOSE: To study the ability of a novel bovine serum albumin-angiotensin II (BSA-Ang II) conjugate to effect responses of the AT1 angiotensin II receptor subtype mediated by the G-protein-coupled and the beta-arrestin pathways. METHODS: Angiotensin II (Ang II) was conjugated with bovine serum albumin and compared with Ang II for competition binding to AT1 receptors, to stimulate aldosterone release from adrenocortical cells, to promote beta-arrestin binding to AT1 receptors, to promote calcium mobilization, and stimulate drinking of water and saline by rats. RESULTS: The BSA-Ang II conjugate was less potent competing for AT1R binding, but was equally efficacious at stimulating aldosterone release from H295R adrenocortical cells. Both BSA-Ang II and Ang II stimulated calcium mobilization and beta-arrestin binding to AT1 receptors. BSA-Ang II and Ang II stimulated water appetite equivalently but BSA-Ang II stimulated saline appetite more than Ang II. Both BSA-Ang II and Ang II were considerably more potent at causing calcium mobilization than ß-arrestin binding. CONCLUSIONS: Addition of a high molecular weight molecule to Ang II reduced its AT1 receptor binding affinity, but did not significantly alter stimulation of aldosterone release or water consumption. The BSA-Ang II conjugate caused a greater saline appetite than Ang II suggesting that it may be a more efficacious agonist of this beta-arrestin-mediated response than Ang II. The higher potency calcium signaling response suggests that the G-protein-coupled responses predominate at physiological concentrations of Ang II, while the beta-arrestin response requires pathophysiological or pharmacological concentrations of Ang II to occur.

FGF23 is endogenously phosphorylated in bone cells.

Levels of serum phosphate are controlled by the peptide hormone FGF23, secreted from bone osteocytes. Elevated levels of circulating FGF23 are a key factor in several hypophosphatemic disorders and play a role in chronic kidney disease. Posttranslational processing of FGF23 includes multi-site O-glycosylation, which reduces intracellular cleavage by proprotein convertases. The FGF23 protein also contains four serine phosphorylation consensus sequences (S-X-D/E); in this work, we asked whether FGF23 is a substrate for secretory phosphorylation. Both HEK cells as well as IDG-SW3 cells, an osteocyte model, incorporated radiolabeled orthophosphate into intact FGF23, as well as into the 14-kDa carboxy-terminal-but not the 17-kDa N-terminal-fragment. Sequential serine-to-alanine site-directed mutagenesis of four kinase consensus sites showed that labeling occurred on three serines within the carboxy-terminal fragment, Ser180 (adjacent to the cleavage site), Ser207, and Ser212. Liquid chromatography-coupled mass spectroscopy indicated the presence of phosphate at Ser212 in recombinant R&D mouse FGF23(R179Q) , confirming labeling results. A phosphopeptide-specific antibody was raised against phospho-Ser212 and exhibited immunoreactivity in osteocytes present in mouse long bone, providing further evidence that FGF23 is naturally phosphorylated in bone. Bone SIBLING proteins are serine-phosphorylated by the ubiquitous Golgi secretory kinase FAM20C. Cotransfection of HEK and MC3T3 cells with FGF23 and active, but not inactive, FAM20C kinase increased the storage and release of FGF23 in radiolabeling experiments, indicating potential effects of phosphorylation on FGF23 stability. Collectively, these data point to an important role for phosphorylation of FGF23 in bone.