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Titin-cap associates with, and regulates secretion of, Myostatin
Gina Nicholas1, Mark Thomas, Brett Langley
1Animal Genomics, AgResearch, East Street, Hamilton, New Zealand.
Abstract:
Myostatin, a secreted growth factor, is a key negative regulator of skeletal muscle growth. To identify modifiers of Myostatin function, we screened for Myostatin interacting proteins. Using a yeast two-hybrid screen, we identified Titin-cap (T-cap) protein as interacting with Myostatin. T-cap is a sarcomeric protein that binds to the N-terminal domain of Titin and is a substrate of the titin kinase. Mammalian two-hybrid studies, in vitro binding assays and protein truncations in the yeast two-hybrid system verified the specific interaction between processed mature Myostatin and full-length T-cap. Analysis of protein-protein interaction using surface plasmon resonance (Biacore, Uppsala, Sweden) kinetics revealed a high affinity between Myostatin and T-cap with a KD of 40 nM. When T-cap was stably overexpressed in C(2)C(12) myoblasts, the rate of cell proliferation was significantly increased. Western analyses showed that production and processing of Myostatin were not altered in cells overexpressing T-cap, but an increase in the retention of mature Myostatin indicated that T-cap may block Myostatin secretion. Bioassay for Myostatin confirmed that conditioned media from myoblasts overexpressing T-cap contained lower levels of Myostatin. Given that Myostatin negatively regulates myoblast proliferation, the increase in proliferation observed in myoblasts overexpressing T-cap could thus be due to reduced Myostatin secretion. These results suggest that T-cap, by interacting with Myostatin, controls Myostatin secretion in myogenic precursor cells without affecting the processing step of precursor Myostatin.
Insights
Titin-cap (T-cap) interacts with myostatin, a protein that limits muscle growth. This interaction appears to reduce myostatin secretion, potentially enhancing muscle cell proliferation.
Area of Science:
- Muscle biology
- Protein interactions
- Cell signaling
Background:
- Myostatin is a critical negative regulator of skeletal muscle mass.
- Identifying proteins that modify myostatin function is key to understanding muscle growth regulation.
Purpose of the Study:
- To identify proteins that interact with myostatin.
- To investigate the functional consequences of myostatin-protein interactions on myoblast proliferation and myostatin secretion.
Main Methods:
- Yeast two-hybrid and mammalian two-hybrid screens to identify interacting proteins.
- In vitro binding assays and surface plasmon resonance to characterize protein interactions.
- Stable overexpression of Titin-cap (T-cap) in C(2)C(12) myoblasts.
- Western blot analysis and bioassays to assess myostatin levels and secretion.
Main Results:
- Titin-cap (T-cap) was identified as a myostatin-interacting protein with high affinity (KD of 40 nM).
- Overexpression of T-cap in myoblasts significantly increased cell proliferation.
- T-cap overexpression led to increased retention of mature myostatin, suggesting blocked secretion.
- Conditioned media from T-cap overexpressing cells showed reduced myostatin levels.
Conclusions:
- Titin-cap (T-cap) interacts with myostatin and modulates its function.
- T-cap controls myostatin secretion from myogenic precursor cells without affecting myostatin processing.
- The interaction between T-cap and myostatin may represent a novel mechanism for regulating skeletal muscle growth.