CONTROL OF CELLULAR PROTEOLYSIS

Project: Research project

Project Details

Description

In this proposed study, we will test the hypothesis that the
degradation of proteins by the ubiquitin-dependent pathway requires
the intermediate formation of a unique multiubiquitin chain on a
substrate protein. In this multiubiquitin chain, the C-terminal
carboxyl group of one ubiquitin molecule is linked via an
isopeptide bond to the e-amino group of lysine 48 of an adjoining
ubiquitin molecule. Whether the degradation of a specific protein
in the in vitro reticulocyte lysate system requires such a
multiubiquitin chain intermediate can be tested initially by
replacing wild-type ubiquitin in the degradative assay with a
ubiquitin mutant, Ub-R48, in which lysine 48 was replaced by an
arginine. Further structural studies can then be made to confirm
the existence of this unique multiubiquitin chain in the
degradative intermediates. The expression of Ub-R48 in the yeast
Saccharomyces cerevisiae will also permit the testing of this
hypothesis in vivo. In order to test the function of this
multiubiquitin chain, we have also proposed to synthesize
chemically a sufficient amount of an analog so that we may study
its interaction with a purified protease which has been shown to
act preferentially on ubiquitinated proteins. Positive results
from these proposed studies should elucidate the mechanism by which
proteolytic targeting is achieved by the posttranslational addition
of ubiquitin to proteins.

We also propose to characterize systematically the
posttranslational addition of ubiquitin to proteins in the yeast
nucleus. These characterizations will include the identification
of nuclear ubiquitin conjugation enzymes and their substrates.
Long term objectives in this research will include the cloning of
the substrate proteins and studies of their functions by gene
deletion and mutations.

As part of our continuing effort to understand our finding of
ubiquitin in the neurofibrillary tangles of Alzheimer's brain, we
wish to determine how an insufficient level of cellular ubiquitin
may cause cell death. Because the ubiquitin system is highly
conserved among eukaryotes, relevant biochemical changes caused by
an insufficient level of ubiquitin can be determined using the
yeast Saccharomyces cerevisiae. Studies are proposed here to
characterize the biochemical changes in a yeast mutant in which the
polyubiquitin gene had been deleted.
StatusFinished
Effective start/end date12/31/896/30/93

Funding

  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences

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