Structural determinants and genetic modifications enhance BMP2 stability and extracellular secretion

The short half-life and use of recombinant bone morphogentic protein (BMP)-2 in large doses poses major limitations in the clinic. Events regulating post-translational processing and degradation of BMP-2 in situ, linked to its secretion, have not been understood. Towards identifying mechanisms regulating intracellular BMP-2 stability, we first discovered that inhibiting proteasomal degradation enhances both intracellular BMP-2 level and its extracellular secretion. Next, we identified BMP-2 degradation occurs through an ubiquitin-mediated mechanism. Since ubiquitination precedes proteasomal turnover and mainly occurs on lysine residues of nascent proteins, we systematically mutated individual lysine residues within BMP-2 and tested them for enhanced stability. Results revealed that substitutions on four lysine residues within the pro-BMP-2 region and three in the mature region increased both BMP-2 turnover and extracellular secretion. Structural modeling revealed key lysine residues involved in proteasomal degradation occupy a lysine cluster near proprotein convertase cleavage site. Interestingly, mutations within these residues did not affect biological activity of BMP-2. These data suggest preventing intracellular proteasomal loss of BMP-2 through genetic modifications can overcome limitations related to its short half-life.