Abstract:
The first goal of this study was to evaluate the roles of different intracellular
proteolytic systems in accelerated skeletal muscle protein degradation. Total,
myofibrillar and non-myofibrillar proteolysis were determined in cultured L8/CID
muscle cells. Calpain proteolysis was assessed by over-expressed calpastatin inhibitory
domain (CD). Lysosomal proteolysis was determined in the presence of methylamine.
Ub-proteasome proteolysis was measured by inhibiting proteasome activity with Z-Leu-
Leu-Leu-H (aldehyde) (ZLLL). Accelerated protein degradation, induced by serum
withdrawal, increased myofibrillar and non-myofibrillar protein degradation by 31 and
38%, respectively. Inhibition of all protease systems reduced total protein degradation
by approximately 67%. Ub-proteasome and lysosomal pathways accounted for most
protein degradation (62% and 40%, respectively). The lysosome contributed non-selectively
to the myofibrillar protein (27%) and non-myofibrillar protein (27%)
turnover. The Ub-proteasome system appears mainly responsible for the myofibrillar
pool degradation (10%) and has a slight effect in non-myofibrillar protein degradation
(5%). The calpain system has a slight effect on overall proteolysis (20%). However, the
calpain plays an important role in initiating muscle protein degradation.
Inhibition of the lysosomal and proteasome pathways stabilized tropomyosin,
desmin, filamin, α-actinin, and dystrophin. The accumulated levels of desmin and
dystrophin increased by additional CID overexpression, indicate the coordination of the
protease systems in degradation of individual proteins as well as the role of calpain in
initiating protein degradation.
Numerous studies have shown that calpain cleaved a variety of substrates in
vitro. The second goal of this study was to identify the actual substrates of m-calpain in
skeletal muscle. A dominant negative m-calpain was used as a bait for screening human
skeletal muscle cDNA libraries in the yeast two-hybrid system. Four partial cDNAs
encoding creatine kinase, glycogen phosphorylase, Ca²⁺-ATPase and nebulin-related
protein were identified as m-calpain binding proteins. Purified creatine kinase and
glycogen phosphorylase were cleaved by m-calpain in an in vitro assay. These results
suggest that creatine kinase and glycogen phosphorylase interact with m-calpain and m-calpain
may regulate the degradation of creatine kinase and glycogen phosphorylase
and possibly regulate their activities. To date, we have not yet evaluated whether Ca²⁺-
ATPase and nebulin-related protein also serve as substrates for calpain.
