The first goal of this study was to understand the role of calpains in skeletal muscle
protein degradation in cultured cells. We have developed a genetic approach to inhibit
endogenous calpain activity through over-expressing dominant negative m-calpain (DN),
antisense m-calpain (AS) and calpastatin inhibitory domain (CID). We observed that,
under conditions of accelerated degradation (serum withdrawal), inhibition of m-calpain
through DN-m-calpain over-expression caused a 30% inhibition of total protein
degradation whereas CID over-expression reduced degradation by 63%. These
constructs did not significantly affect degradation in the presence of serum. These data
indicate that calpains participate in the accelerated degradation associated with serum
withdrawal. Inhibition of calpain also stabilized nebulin, a major structural protein of the
sarcomere. These observations indicate that calpains play significant roles in muscle
protein turnover. Finally, over-expression of antisense m-calpain caused a transient
reduction in m-calpain concentration after which normal m-calpain concentration was
quickly re-established. These observations indicate that m-calpain is a short half-life
protein in muscle cells.
The second goal of this study is to investigate the role of calpain in the mediation
of PARP protein level in differentiating myoblasts. Poly(ADP-ribosyl)ation, catalyzed by
PARP, is involved in various physiological events, such as DNA excision repair, DNA
recombination, DNA replication, cell differentiation, cell growth and transformation, and
apoptosis. A protease participating in PARP turnover could be a significant regulator to the events which PARP is involved. A relationship between apoptosis and myofibrillar
protein degradation via a common protease might suggest the basis for muscle wasting
and atrophy which characterize in many muscle diseases. We established a genetic
approach to inhibit endogenous calpain activity through over-expressing calpastatin
inhibitory domain (CID). We observed that (1) inhibition of calpain activity increased
PARP concentration when post-confluent myoblasts were cultured with 2% HS medium,
an inducer of differentiation and (2) inhibition of calpain activity prevented PARP
degradation induced by A23187 and etoposide in differentiating myoblasts. These data
demonstrate that calpain is involved in regulation of PARP in cultured cells. / Graduation date: 1998
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/33998 |
| Date | 13 April 1998 |
| Creators | Huang, Jing, 1961- |
| Contributors | Neil E., Forsberg |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
Page generated in 0.0046 seconds