107 A Clinically Important Post-Translational Modification of CTRP9: Proteolytic Cleavage of CTRP9 Yields its Globular Domain, Which Activates AMPK

Abstract

C1q/tumor necrosis factor-related protein-9 (CTRP9) is a newly identified adiponectin (APN) paralog with metabolic regulatory properties. We previously demonstrated CTRP9 attenuates adverse cardiac remodeling after acute myocardial injury. From its protein structure, CTRP9 is predicted to possess a globular domain (similar to APN). However, whether the globular domain of CTRP9 (g-CTRP9) exists physiologically in cells is completely unknown. This study determined whether CTRP9 undergoes in vivo proteolytic cleavage, and its resultant functional significance. In mouse serum, Western blot analysis employing an anti-CTRP9 antibody consistently reveals 2 solid bands (42 KD, the predicted weight of full-length CTRP9 [f-CTRP9], and 17 KD, the predicted weight of g-CTRP9). Whereas CTRP-9 expressed by HEK293T cells also exhibits both 42 and 17 KD bands, only f-CTRP9 is observed by Western blot of bacteria-generated CTRP9, suggesting CTRP9 processing requires a eukaryotic mechanism. To confirm this conjecture, bacteria-produced fCTRP9 was incubated in an environment of lysed HEK293 cells. Subsequent Western analysis revealed both f- and g-CTRP9. To give further evidence of proteolytic cleavage of CTRP9, we employed genetic manipulation techniques, inserting a Flag tag at the N-terminus and a Myc tag at the C-terminus of CTRP9 produced by both 3T3-L1 and HEK293 cells. Utilizing only Flag and Myc antibodies, we confirm CTRP9 is cleaved and gCTRP9 is produced in both 3T3-L1 and HEK293 cells. Finally, we determined the functional significance of CTRP9 cleavage. H9C2 cells were starved and subjected to treatments of either f- or g-CTRP9 for 1 hour. f-CTRP9 decreased AMPK activation, whereas g-CTRP9 increased AMPK activation, as determined by Western blot assessment of ACC, pACC, and pAMPK. We demonstrate full length CTRP9 is subjected to in vivo proteolytic cleavage, yielding its globular domain (the main circulatory form of CTRP9). More importantly, only g-CTRP9 increased AMPK activation, which is clinically important, because AMPK activation is beneficial in the setting of ischemia/reperfusion injury as well as metabolic disorders. Further studies specifically determining the mechanisms underlying the proteolytic cleavage of f-CTRP9 remain ongoing.