Within the folds, outside the box
Susan Lindquist uncovers the roles that misshapen
proteins play across an astonishing sweep of phenomena
Feverishly hot climates. Dizzying alcohol and sugar
binges. Heavy metals. Toxic drugs. Genetic mutations.
Over the years, yeast, fruit flies, mustard plants and
mice have struggled through their own versions of an
extreme reality TV show in the laboratory of Whitehead
Member Susan
Lindquist.
The challenges to these different critters have been
limited only by the overactive imaginations of Lindquist
and her colleagues. One recent episode involved forcing
yeast cells to evolve resistance to the best drugs used
in clinics to fight deadly fungal infections. In future
installments, the yeast must first imitate and then
overcome a protein problem underlying a horrific human
neurodegenerative disease.
The amazing but true stories of how some of the yeast
have endured, and even thrived, have advanced a strangely
wide array of science: mad cow disease, neuroscience,
nanotechnology, cancer, anti-fungal drug resistance
and non-genetic evolution.
But these seemingly disparate discoveries share a common thread.
"The one universal theme in our lab is protein folding
and how changes in protein folding drive many biological
processes," explains Lindquist in her sixth-floor office
overlooking MIT on a hazy day.
"People didn't realize how broad the protein folding
problems are. A lot of things that started out as basic
research into protein folding are now translating into
a direct interest in human health and medicine."
Researchers in her lab have even found that proteins
can trump DNA in passing along new traits into the genomes
of future generations.
"You don't usually think about proteins this way,"
acknowledges James Shorter, a senior research associate.
"Independent of the underlying DNA, protein folding
can influence a wide variety of things, from evolution
to disease progression and initiation. And it can act
as a genetic element. Initially, this seemed just crazy,
but it is true."
| "Protein folding is deeply rooted in biology,"
Lindquist says. "All organisms face the same problems
and share the same solutions."
|
Protein pirouettes
Proteins start as linear strings of building blocks—assorted
combinations of 20 amino acids specified by the genetic
blueprint. Some strings are short. Some are long. All
must fold into complex shapes to do their jobs. The
shape of a protein gives it its function.
Less than a minute after they are formed, proteins
loop, twist and scrunch themselves into neat preordained
packages featuring lobes, spirals, pleats and hinges.
The nascent protein attempts its precision maneuvers
in a tightly packed cell, bumped and jostled by hundreds
of other proteins hustling and bustling about the cell.
It is as if everyone on a crowded bus started doing
aerobics all at once, each to his or her own style,
tune and timing.
Not surprisingly, nearly one-third of proteins cannot
fold properly, Lindquist says. The problem could be
the molecular equivalent of a knee to the gut, or it
might have been a nick or ding in the original genetic
information that miscued a crucial nook or cranny.
A distorted protein may be unable to carry out its
crucial mission, or it may have transformed into something
nasty. Either way, "it can be an absolute disaster,"
Lindquist says. "Misfolded proteins are responsible
for many terrible illnesses of mankind. In cystic fibrosis,
just one amino acid in several hundred is wrong. This
means that this one protein can't quite fold up properly
to get to the surface and function. Disaster." Another
variation of misshapen proteins are amyloids, nearly
indestructible amalgamations of proteins found in Alzheimer's,
Huntington's and Parkinson's diseases.
Surprisingly, her lab is finding that in some circumstances,
an alternative fold in a protein may underlie vital
aspects of normal biology.
A model menagerie
Lindquist, who started as a fruit fly cell biologist,
now tracks warped proteins and their consequences through
model systems spanning millions of years of evolution
She switched to yeast as her main model 22 years ago
after attending the Cold Spring Harbor Yeast Genetics
Course co-led by Whitehead Founding Member Gerald
Fink. But the lab nimbly moves through an experimental
menagerie that also includes plants, mice and human
cancer cell lines. In collaborations with others, the
team has added expertise in rats and sea slugs.
For example, in a sea slug's oversized neurons, the
protein that sustains long-term memory at the junctions
and synapses seems to work by shifting its shape into
a prion, a configuration that bends other proteins to
its same altered form.
This finding, reported two years ago in collaboration
with neurobiologist Eric Kandel at Columbia University,
complemented unexpected aspects of prion activity in
yeast.
It contradicted a widely held belief that prion activity
is inevitably toxic, a generalized assumption inspired
by the well-publicized and frightening cases of mad
cow disease transmitted to people.
And the slug protein work suggested an unexpected new
mechanism for long-term memory in higher organisms as
well.
"Protein folding is deeply rooted in biology," Lindquist
says. "All organisms face the same problems and share
the same solutions. Mother Nature has been coping with
protein folding problems since the dawn of time. It
makes sense that she would discover ways to turn it
to her advantage."
Heat shock and awe
Potentially devastating protein folding problems worsen
when a cell or organism is stressed by hostile environmental
factors, such as heat. In response, cells send in a
rescue squad called heat shock proteins, also known
as chaperones, to resuscitate or cart away proteins
missing their full complement of nips and tucks.
Some of the emergency workers prevent unfolded proteins
from aggregating. Some disaggregate dysfunctional clumps.
Some hold proteins in partially folded states until
they receive the right signal, perhaps from a hormone.
Some act as trash collectors for the irredeemably malformed.
Others refold wilted proteins and give them a second
chance.
In a sense, heat shock proteins also have chaperoned
Lindquist's career, beginning with her independent decision
to study them in fruit fly tissue culture when she was
a graduate student in the Harvard lab of Matthew Meselson.
Then, she was more interested in the rapid change in
gene expression patterns stimulated by environmental
stress, which is anything but subtle. Indeed, it is
a cellular shock and awe tactic, with genes churning
out 50 to 10,000 times more heat shock proteins to try
to save a cell from its environment.
At the University of Chicago, where she did her postdoctoral
work and progressed to full professor and Howard Hughes
Medical Institute investigator, Lindquist helped unmask
the function of heat shock proteins in the protein folding
response.
Back in her fruit fly days, Lindquist and her collaborators
had figured out that one known heat shock protein, Hsp90,
worked by helping proteins with minor mutations that
would otherwise alter their form. Taking away Hsp90
unveiled complete sets of hidden mutations with new
functions. If the new variations were advantageous,
Lindquist's team showed, the breeding flies' offspring
hung onto the helpful mutations and the remodeled proteins,
even without the assistance of Hsp90.
Three years ago, Lindquist's group announced that Hsp90
performs the same trick in the experimental plant thale
cress, making it likely that other organisms can also
save up genetic changes for a rainy day. The mechanism
could help to explain some of the rapid diversification
found in the fossil record.
On a darker note, the group also has
found that Hsp90 enables mutations in human cancer cells
to promote cancer growth. In animals, Hsp90 inhibitors
can reverse oncogenic transformations and are now in
clinical trials.
Most recently, postdoctoral researcher Leah Cowen has
shown that Hsp90 allows yeast to rapidly evolve resistance
to antifungal drugs. Once resistance has evolved, compromising
Hsp90 functions with drugs or mutations can abolish
it. Remarkably, yeast exposed to temperatures that simulated
human fevers lost drug resistance, mimicking the effects
of inhibiting Hsp90 function. This provides one of the
first molecular explanations of a beneficial role for
fevers.
Protein busters
In yeast, Lindquist discovered another member of the
heat shock family, Hsp104. This protein proved to be
a powerful protein remodeling agent that saved yeast
from sudden high-temperature heat shocks and all sorts
of other stressful environmental conditions her lab
could conjure.
And it seemed to be doing the impossible. Unlike Hsp90,
which holds onto proteins and prevents them from misfolding,
Hsp104 works to take apart proteins that have aggregated
together. That finding reversed a common dogma that
misfolded and aggregated proteins are irredeemable,
Lindquist said. Instead, the chaperone rescues congealed
proteins and restores them to their individual functions.
"You can't unfry an egg, but you can uncoddle an egg,"
she says.
Strikingly, Hsp104 can also pass along and release
hidden genetic variation. As part of normal yeast biology,
Hsp104 remodels a protein named Sup35 into a prion named
[PSI+]. Lindquist's team showed that Hsp104 was necessary
to refold Sup35, but once transformed, [PSI+], a regulator
of protein synthesis, is positively evangelical about
converting other Sup35 proteins to the same altered
shape. Hsp104 ensures that mother cells pass along the
prion to daughter cells, whose proteins are thereby
influenced to keep changing shape, too.
This goes on for generation after generation.
Why would cells have a protein that changes shape like
this? [PSI+] removes the stop sign that normally appears
when proteins are being synthesized: Ribosomes roll
through their normal stopping point on an RNA strand
and read into fresh genetic regions. Many proteins are
outfitted with extra features, which may provide a survival
advantage in a fluctuating environment and thus eventually
become genetically fixed.
Perplexingly, the prions created by low levels of Hsp104
can be disaggregated by high levels of Hsp104. Last
year, Shorter helped resolve this major conundrum, publishing
in the journal Science. Shorter worked out
the complicated and dramatically shifting biochemistry
by mixing the two proteins Hsp104 and Sup35 in a test
tube with various sources of energy.
"This is the first time that anyone has found anything
that can catalytically take apart an amyloid fiber,"
Lindquist says. In the lab, protein amyloids, like those
that clog up the brains of people who died from Alzheimer's
disease, are impervious to just about anything, including
extreme heat and cold and powerful detergents.
The yeast prion amyloid fibers are also remarkably
resilient, able to withstand exposure to extended high
temperatures, high and low salt, strong alkalis and
acids, and 100 percent ethanol.
Before coming to Whitehead, in fact, Lindquist and
her collaborators at the University of Chicago exploited
the strength of these protein-protein connections to
make nanoscale wires of gold and silver a thousandth
the thickness of a human hair that successfully conducted
electricity.
In the last few billion years, animal cells lost the
ability to produce Hsp104. "You can imagine how it might
be useful for diseases associated with protein aggregation,"
Shorter says. "If we understand how it works, we can
apply it to other systems."
The mad cow connection
The helpful yeast prion protein and the dangerous mammalian
prion protein have virtually nothing in common. But
the ability of [PSI+] to self-replicate by changing
the shape of other proteins is eerily similar to the
way the infectious mad cow protein seems to corrupt
a plentiful membrane protein in people's brains into
an insidious shape that causes horrific disease. And
the two proteins have one vaguely similar region.
Further experiments in yeast and mice along these lines
led Lindquist to propose a new, unifying hypothesis
to explain the origin of the human prion disease and
the mechanism of its toxicity.
Bits of misfolded proteins processed by specialty organelles
may accumulate in the main compartment of the cell,
the cytosol, where they can be tagged for disposal by
the cellular garbage service. The volume may cross a
threshold, where the cell's quality control systems
cannot remove the misfits fast enough. Even a barely
detectable level of misshapen proteins can be toxic
to a neuron.
Probing Parkinson's
Using the yeast as a living test tube, a team led by
graduate student Tiago Outeiro has showed that overproduction
of a human protein, alpha-synuclein, can convince neighboring
proteins to abandon their normal shape and form protein
clusters similar to those in Parkinson's disease. The
afflicted yeast suffer from a similar range of symptoms
and die.
"We have reason to believe it is a quality control
problem," Lindquist says. “In some people, the protein
misfolds at a higher rate, and that becomes a disaster
in a hurry. In other people, as they age, the protein
folding quality control system gets wimpy and can't
keep up with the normal rate of misfolding."
Her team screened 116,000 chemical compounds to reverse
the toxicity of alpha-synuclein overload in yeast. Among
the 60 compounds, they found one that previously had
been used as an antibiotic and is now in clinical trials
for Alzheimer's disease. "That makes me think we've
found something real," Lindquist says. "We hope we will
be able to develop therapeutic strategies in yeast."
Postdoctoral fellow Aaron Gitler now is searching for
the original defect that the Parkinson's protein triggers
in yeast cells in hopes of identifying the underlying
disease pathway and key drug targets.
Out of boundaries
Not surprisingly, Lindquist can't predict where this
rich and deep collection of studies will lead her.
"I hope it won't be something I anticipate now," she
says. "Seventy percent of what I'm now doing I couldn't
have foreseen five years ago.
"It happens in other labs too. You take unexpected
twists and turns not only from your own data but from
responding to the scientific community at large."
Whatever the future brings, Lindquist is likely to
be more closely involved in human diseases. Last year,
she co-founded FoldRX Pharmaceuticals, which will develop
drugs to treat diseases of protein misfolding. She also
was elected to the board of directors for Johnson &
Johnson.
"Susan has enormous creativity," says close friend
Elaine Fuchs, a Howard Hughes investigator at Rockefeller
University and a member of Whitehead's Scientific Advisory
Board. "Her ability and vision to think about areas
of science so broadly allow her to make connections
that are quite extraordinary and lead to interesting
science." Those significant connections extend beyond
science, adds Fuchs, whose marriage resulted from Lindquist's
penchant for matchmaking.
Lindquist puts it another way. "There's a great deal
to be said for concentrating on one thing," she says.
"I'm the exact opposite."
|
