CONTROL OF CELLULAR PROTEOLYSIS

Project: Research project

Description

Proteins to be degraded via the cytoplasmic ubiquitin-dependent pathway are
first covalently linked with ubiquitin. These ubiquitin-protein conjugates
are then recognized and degraded by the proteolytic component of the
pathway. One of the major thrusts of this proposed research is to identify
this proteolytic component and to characterize the proteolytic reaction
using ubiquitin-calmodulin as a substrate. In order to determine how an ubiquitin-protein conjugate is recognized and
degraded by the proteolytic component of the ubiquitin dependent pathway,
it requires a substrate having a known structure, and for which the
proteolytic products can be characterized. Dictyostelium calmodulin forms
a one-to-one conjugate with ubiquitin and specifically at lysine 115. All
other substrate proteins reported to date are conjugated with ubiquitin at
multiple sites, and the conjugation sites in these proteins have yet to be
defined. Using ubiquitin-calmodulin as a substrate, we have tentatively
identified an ATP-dependent protease in reticulocytes which degrades
calmodulin only in the conjugated form. The proposed studies include the
continued purification of this protease and to characterize the cleavage of
ubiquitinated calmodulin. Synthetic peptides containing the isopeptide
linkage, as well as small calmodulin fragments conjugated to ubiquitin,
will be tested as potential substrates or as inhibitors. These results,
together with the mapping of the cleavage sites of conjugated calmodulin,
will be used to test the hypothesis that the isopeptide linkage region
serves as the recognition site for the protease. Calmodulin isolated from most sources contains a trimethyllysine at
position 115 and cannot be conjugated with ubiquitin. Our current
hypothesis is that the post-translational methylation of this lysine
stabilizes this protein by excluding it from degradation via the
ubiquitin-dependent pathway. This hypothesis will be tested by measuring
the degradative rates of various forms of microinjected calmodulin in
oocytes under normal and under conditions in which the ubiquitin-dependent
pathway has been rendered inactive. We will also test whether acetylation
of the N-terminus of calmodulin serves the same function by using bacterial
expressed chicken calmodulin which contains an unblocked N-terminus.
StatusFinished
Effective start/end date7/1/866/30/93

Funding

  • National Institutes of Health
  • National Institutes of Health: $134,206.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

Fingerprint

ubiquitin
proteolysis
calmodulin
Ubiquitin
Proteolysis
proteinases
proteins
Proteins
Lysine
Ubiquitinated Proteins
Neurofibrillary Tangles
Dictyostelium
Reticulocytes
reticulocytes
synthetic peptides
Eukaryota
Saccharomyces cerevisiae
Organism Cloning
Cause of Death
methylation