Berkeley researchers have provided the most detailed look ever at the “regulatory particle” used by proteasomes to identify and degrade proteins marked for destruction. The particle is organized into two sub-complexes, a “lid” and a “base.” |
Lawrence Berkeley
National Laboratory researchers have provided the most detailed look ever at the
“regulatory particle” used by proteasomes to identify and degrade
proteins marked for destruction. The particle is organized into two
sub-complexes, a “lid” and a “base.”
Important
new information on one of the most critical protein machines in living
cells has been reported by a team of researchers with the U.S.
Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley
Lab) and the University of California (UC) Berkeley. The researchers
have provided the most detailed look ever at the “regulatory particle”
used by the protein machines known as proteasomes to identify and
degrade proteins that have been marked for destruction. The activities
controlled by this regulatory particle are critical to the quality
control of cellular proteins, as well as a broad range of vital
biochemical processes, including transcription, DNA repair and the
immune defense system.
“Using
electron microscopy and a revolutionary new system for protein
expression, we have determined at a subnanometer scale the complete
architecture, including the relative positions of all its protein
components, of the proteasome regulatory particle,” says biophysicist
Eva Nogales, the research team’s co-principal investigator. “This
provides a structural basis for the ability of the proteasome to
recognize and degrade unwanted proteins and thereby regulate the amount
of any one type of protein that is present in the cell.”
Says
the team’s other co-principal investigator and corresponding author,
biochemist Andreas Martin, “While the biochemical function of many of
the proteasome components have been determined, and some subnanometer
structures have been identified, it was unclear before now which
component goes where and which components interact with one another. Now
we have a much better understanding as to how the proteasome machinery
works to control cellular processes and this opens the possibility of
manipulating proteasome activity for the treatment of cancer and other
diseases.”
Nogales,
who holds appointments with Berkeley Lab, UC Berkeley, and the Howard
Hughes Medical Institute, and Martin, who holds appointments with UC
Berkeley and the QB3 Institute, are the senior authors of a paper
describing this work in the journal Nature.
The paper is titled “Complete subunit architecture of the proteasome
regulatory particle.” Other co-authors were Gabriel Lander, Eric Estrin,
Mary Matyskiela and Charlene Bashore.
At
any given moment, a human cell typically contains about 100,000
different proteins, with certain proteins being manufactured and others
being discarded as needed for the cell’s continued prosperity. Unwanted
proteins are tagged with a “kiss-of-death” label in the form of a
polypeptide called “ubiquitin.” A protein marked with ubiquitin is
delivered to any one of the some 30,000 proteasomes in the cell—barrel-shaped complexes which act as waste disposal units that rapidly
break-down or degrade the protein. The 2004 Nobel Prize in chemistry was
awarded to a trio of scientists who first described the proteasome
process, but a lack of structural information has limited the scientific
understanding of the mechanics behind this process.
Nogales,
an expert on electron microscopy and image analysis, and Martin, who
developed the new protein expression system used in this work, combined
the expertise of their respective research groups to study the
proteasome regulatory particle in yeast. The particle features 19
sub-units that are organized into two sub-complexes, a “lid” and a
“base.” The lid contains the regulatory elements that identify the
ubiquitin tag marking a protein for destruction, and the base features a
hexameric ring that pulls the tagged protein inside the chamber of the
proteasome barrel where it is degraded.
“The
lid consists of nine non-ATPase proteins including ubiquitin receptors
that accept properly tagged proteins but prevent a protein not marked
for degradation from engaging with the proteasome,” Nogales says. “Since
degradation is irreversible, it is critical that only ubiquitin-tagged
proteins engage the proteasome. Interestingly, the ubiquitin tag has to
be removed before the protein can be translocated into the proteasome’s
destruction chamber, so the lid also contains de-ubiquitination enzymes
that remove the tags after the protein has engaged with the proteasome.”
The
proteasome regulatory particle’s base contains six distinct AAA+
ATPases that form the hetero-hexameric ring, which serves as the
molecular motor of the proteasome.
“We
predict that the ATPases use the energy of ATP binding and hydrolysis
to exert a pulling force on engaged proteins, unfolding and
translocating them through a narrow central pore and into the
degradation chamber,” Martin says. “The steps in the proteasome
process—from protein recognition to de-ubiquitination and degradation
have to be very highly coordinated in time and space. Locating all of
these components and identifying their relative orientations has been
very telling about how the processes are coordinated with each other.”
Nogales
credits the protein expression system developed by Martin and his
research group, in which proteins are expressed and assembled in
bacteria, as being critical to the success of this research.
“Until
now researchers had to work with purified protein complexes from the
cell, which could not be manipulated or modified in any way,” she says.
“Andy Martin’s new heterologous expression system allows for the
manipulation and dissection of protein functions. For our studies it was
crucial to generate lid sub-complexes that had one marker at a time in
each of the subunits so that we could determine the position of each
protein within the lid. With this new system we generated truncations,
deletions and fusion constructs that were used to localize individual
subunits and delineate their boundaries within the lid.”
This
research was supported by funds from UC Berkeley, Berkeley Lab, the
National Institutes of Health, the Searle Scholars Program, the Damon
Runyon Cancer Research Foundation, the American Cancer Society, the
National Science Foundation and the Howard Hughes Medical Institute.
Eva Nogales and her research group
