Pennsylvania University. US: Workhorse molecules called heat-shock proteins contribute to
refolding proteins that were once misfolded and clumped, causing such
disorders as Parkinson's disease, amyotrophic lateral sclerosis, and
Alzheimer's disease. James Shorter, PhD, an associate professor of Biochemistry and Biophysics, at the Perelman School of Medicine at the University of Pennsylvania,
has been developing ways to "reprogram" one such protein – a yeast
protein called Hsp104 -- to improve its therapeutic properties.
But precise knowledge about the mechanisms by which Hsp104 works
to fix misshapened and clumped proteins has been lacking. Now, Shorter
and his colleagues have discovered that a previously disregarded part
of the Hsp104 structure, the N-terminal domain (NTD), located at one
end of the Hsp104 molecule, is a major player in its protein-busting
powers. Their work was published in Molecular Cell.
"We've defined in unprecedented detail the mechanism by which
Hsp104 dissolves its natural substrate, Sup35 prions," says Shorter.
"We found that the N-terminal domain of Hsp104 allows the enzyme to
function in a way that enables the disintegration of the prion." Prions
are “infectious” proteins that cause disease in humans, but can be
beneficial in yeast.
While Hsp104 is found in the vast majority of less complex
organisms on the planet, it was somehow lost in the evolution of lower
forms of life to more complex animals and humans. “It's baffling in
many ways," says Shorter. "We don't understand quite why Hsp104 was
lost. But it could be useful in a therapeutic setting because we could
add back an activity that humans don't really have: the ability to
rapidly dissolve and refold prions." Previously, Shorter and colleagues
defined a set of human heat shock proteins that can slowly dissolve prions.
Although previous work by Shorter and others had shown that the
middle section of Hsp104 was vital for its clump-busting activity, the
N-terminal domain was thought to be relatively unimportant.
"Researchers had thought it was a more dispensable domain," says lead
author, Elizabeth Sweeny, PhD, a former graduate
student in the Shorter lab who is now a postdoctoral fellow at the
Massachusetts Institute of Technology. "We reveal in this paper that
when you give Hsp104 a very difficult protein clump to break up, like
those seen in neurodegenerative disease protein inclusions, it actually
becomes very important."
Shorter and his collaborators used small-angle X-ray scattering
(SAXS) to examine the role of the Hsp104 N-terminal domain by deleting
it from the enzyme and testing it under different conditions. When
Hsp104 lacking the NTD (Hsp104∆N) is introduced into the formation of
the Sup35 prions in a test tube, it promoted prion formation, instead
of solubilizing prions. The researchers also observed that, while
Hsp104 attacks the Sup35 prion by breaking up the head and tail contacts
that hold the prion together, Hsp104∆N was unable to do likewise.
Hsp104∆N is able to dissolve disordered protein aggregates but cannot
break down prions due to their increased stability.
Sweeny found that Hsp104 – normally shaped like a short, hexagonal tube -- works
like a peristaltic pump that shuttles molecules through its central
channel. ATP, the cell’s energy molecule, is the fuel that powers the
pump.
The altered structure of Hsp104∆N greatly impairs this normal
mechanism, affecting its ability to break apart Sup35 and other prions.
Sweeny notes, "Hsp104 extracts individual proteins from the prion
fibril by pumping them through its central channel and that's how it
dissolves them. The N-terminal domain of Hsp104 allows the enzyme to
function in a more powerful way that enables dissolution of the very
stable Sup35 yeast prion."
These new insights into the workings of Hsp104 open up a range of
new research directions: "We've advanced to a new level of
understanding that will help us design and engineer the enzyme to work
better against the human proteins that are causing issues in disease,"
Shorter notes. “Our next step will be to achieve even greater insight
into Hsp104 structure. Our level of structural resolution is only about
7 or 8 angstroms at the moment and we'd like to get that down to the
atomic level. Another goal is to engineer the N-terminal domain to work
better against clumped human disease proteins. Prior to this paper, we
wouldn't even have thought about doing that because the domain was not
considered important. That’s one of the key findings from this study."
The work was supported by American Heart Association predoctoral
and postdoctoral fellowships; NIGMS, NINDS, and NIH Common Fund grants
(T32GM008275, T32GM071339, F31NS079009, DP2OD002177, R01GM099836), an
Ellison Medical Foundation New Scholar in Aging Award, Target ALS,
Muscular Dystrophy Association (MDA277268), and The Robert Packard
Center for ALS Research at Johns Hopkins University.