The Case for Activity-Based Proteomics
By Jason Socrates Bardi
No doubt owing to the success and hype of the Human Genome Project, several
other "'omes"—the glycome, the transcriptome, the proteome—have
been popping up in the English lexicon, like so many decorative statues
on a green lawn.
These other 'omes are no mere ornaments, though. As scientific endeavors
go, unraveling the secrets of the carbohydrate in the human glycome, the
proteins in the human proteome, and the mRNA in the human transcriptome
follow naturally from the human genome. In fact, understanding them may
even be necessary for the realization of the promise of the human genome—that
mapping will reveal much about human biology, including possible targets
and tools for addressing human disease.
Perhaps the most immediately important of these post-genome 'omes is
the proteome, which by some estimates is at least an order of magnitude
more vast than the human genome. With multiple, alternative RNA splices,
post-translational modifications, and protein folding controlled by localization,
protein interactions, cofactors, temperature, pH, and a dozen other factors,
the 40,000 or so genes in the human genome multiply into millions of possible
protein forms and states in the cell.
What is really interesting is to look at a portion of the proteome—those
gene products that are specifically involved in some discrete pathology.
What could offer more insight than looking at the protein "profile" of
a cancerous cell compared to a normal cell, of a cancerous cell soaked
in a drug compared to one that is not?
These are applications within the scope of proteomics, which attempts
to elucidate the actual expression and action of the genome in different
cell types and states. One important aspect of this is where and when
the proteins are working in the human body, and how they are turned on
or shut off in certain cell types, times, or conditions like infection
or cancer.
A new proteomics technology, which is being developed and applied by
two investigators at The Scripps Research Institute (TSRI) seeks to answer
an even more profound question—which proteins are active in
a given cell, tissue, or disease state?
"It's a chemical approach to understanding protein function," says Cell
Biology Associate Professor Benjamin Cravatt, who began developing the
approach about three years ago. Soon afterwards, he began collaborating
with his colleague and fellow investigators in the Skaggs Institute for
Chemical Biology, Chemistry Associate Professor Erik Sorensen.
The Problem With Proteomics
Generally, proteomics is still an application in search of its technology.
While there are powerful methods for the global analysis of mRNA levels
in cells based on gene chip technology. There are also tools for looking
at proteins—using two-dimensional electrophoresis coupled with mass
spectrometry, for instance. These techniques have lacked luster when it
comes to profiling protein activities, though. Methods that are based
on profiling expression levels may not be sensitive to changes in the
activity of a protein. And methods that do directly look at protein activity
do not necessarily do so in the native state of the cell.
Current proteomics methods may also miss proteins that are only expressed
in low abundance, which may be exactly the proteins that are of interest.
Most of the proteins expressed in a cell, after all, are structural and
housekeeping proteins. And they may miss proteins that are associated
with membranes, proteins with extreme isoelectric points—either very
basic or very acidic proteins—and proteins that have very high or
very low molecular weights.
Cravatt believes that activity-based profiling of proteins expressed
in living cells is the next step
"This is a way to accelerate the process [of finding] active enzymes
that correlate with disease [and] circumvent major challenges that face
proteomics technologies," says Cravatt.
Based on Fundamental Enzymology
The basis of Cravatt's technique is thoroughly grounded in over a half-century
of classical enzymology, the biochemical field concerned with the activity
and rates of catalytic biological molecules.
Over the decades, biochemists have advanced their understanding of various
enzymes by developing chemical probes called "affinity labels." These
are simple, small chemicals with a reactive moiety that has the ability
to attach to the active sites of protein enzymes. These chemical moieties
are used to try to understand the mechanism through which enzymes work.
Active-site proteomics takes this classical approach a step further.
Rather than employing chemical probes to profile one enzyme, the technology
uses probes to assay the activity of entire enzyme families in complex
proteomes. These probes combine a reactive group that binds and covalently
modifies the active sites of many members of a given enzyme class, with
a chemical (e.g., fluorescent) tag for the detection and isolation of
reactive enzymes.
The idea is simple: throw these probes in together with different types
of cells, let them label their enzyme targets, and then do a separation.
Usually this separation involves first splitting cells into the extracellular
fraction (which contains the secreted proteins), the lipidic fraction
(containing the membrane-bound proteins) and the cytosolic fraction (with
the soluble, intracellular proteins). Then the tagged enzymes in each
portion can be further separated and identified by time-tested methods
like running a gel.
By taking these labels and applying them in cases where there is some
phenotype of interest—a metastatic cell, for instance—activity-based
proteomics can give a readout of which proteins in the cell are active
and which are not. Simple.
But nothing is ever that simple.
The real trick is to find the right probes. The best experimental design
is to cover the maximum number of enzymes in a cell with the minimum number
of what they call "promiscuous" probes. The point is not necessarily to
look at the absolute activity of proteins in the mix but to interrogate
broadly and see whether proteins are on or off.
Luckily, when Cravatt started developing the technology, he already
had one such probe—an affinity agent for a family of enzymes called
the serine hydrolases. He had been using the probe in the course of his
studies on the fatty acid amide hydrolase (FAAH) and other members of
the serine hydrolase family. It was known that the FAAH probes were promiscuous
and reacted broadly with other serine hydrolases, and it was a relatively
simple matter to doctor these known affinity labels with readout tags.
"The beauty of it is that it reacts broadly within that family and it
tends not to react with enzymes of other classes," says Sorensen.
So far, they have begun using the technology with this probe to study
melanoma, breast carcinoma, and ovarian carcinoma cells. They are even
able to detect proteins at the femtomole level—as few as a hundred
or so copies of per cell.
Activity-Based Profiling of Cancer Lines
By far the most interesting application of the technology is comparative
profiling, which aims to detect disease-related protein activities and
to establish the identity of proteins that are involved in the pathogenesis
of diseases by comparing, for instance, the active proteins in tumor cells
to normal cells.
In what Cravatt calls the first practical application of the new technology,
a paper in this week's Proceedings of the National Academy of the Sciences
describes the application of the approach to cancerous cells.
Using the serine hydrolase probes, Cravatt and his student Nadim Jessani
looked for proteins that could uniquely identify different cell lines,
like a fingerprint or an eye scan. They chose well-characterized cell
lines to start with, so that they would have a baseline from which to
judge the effectiveness of the technology.
What Jessani and Cravatt found were clusters of proteases, lipases,
and esterases that they could use to distinguish human breast cancer cells
from other types of cancer cells. They also found that they could use
these markers to distinguish invasive tumor cells—those that can
migrate to new tissues after metastasis—from non-invasive ones.
More importantly, they also found that invasive cells from different
cell lines looked more like each other than they do like the non-invasive
cells that they derived from.
"It's not just the upregulation of new enzymes," says Cravatt. "The
cells shut off the original markers that would lead you to believe that
they are, for example, a melanoma."
Furthermore, they identified an enzyme, the membrane-associated hydrolase
KIAA1363, that had previously not been associated with cancer. Based on
the fact that KIAA1363 is up-regulated in invasive cancer cell lines,
Cravatt and Jessani suggest that this enzyme may represent a new marker
of tumor progression.
They were even able to predict invasiveness in other cells based on
the readout of their activity-based assays and the upregulation of KIAA1363.
Significantly, many of the proteins that they were able to profile using
the technology are secreted, found in the membrane, or are expressed in
low abundance—difficult to study using traditional proteomics methods.
"[The paper] shows that we can go into cell lines that have been characterized
for a decade and see new stuff, such as enzymes that correlate with invasiveness
that have never been seen before," says Cravatt.
As insightful as these studies are, they are nevertheless limited by
the breadth of the probe, which has the ability to label all the active
serine hydrolases but none of the active proteins in other families. And
while the serine hydrolases as an incredibly large family, comprising
fully one percent of all predicted gene products, that still leaves the
glass 99 percent empty.
There are many other important families of enzymes that would be interesting
to study using the method, but for which suitable probes are lacking.
The kinases, the metalloproteinases, the phosphatases, for instance, are
all large families with many enzymes relevant to numerous disease pathologies
waiting to be interrogated.
"Can we generalize the concept to address other important enzyme families?"
asks Sorensen. "That is the question."
A More Generalized Approach
It's a question that's simple to pronounce but hard to solve.
Indeed, any chemical that can attach itself to a residue in the active
site of a protein could be a potential probe. The problem is finding ones
that react broadly.
In order to address this, Cravatt, Sorensen, and their student Greg
Adam took what they call a non-directed strategy to come up with more
candidate probes.
They made combinatorial libraries of probes based on a common "chemotype"—sulfonyl
esters, which are commercially available in hundreds of variations, linked
to a variable alkyl/aryl binding group. By attaching different binding
groups to various sulfonyl esters, the nature of the protein activity
profile can be changed. By varying these binding groups, the researchers
constructed a library of compounds and then screened these against complex
proteomes, looking for activity-dependent protein reactivity.
In a Nature Biotechnology article appearing this month, Sorensen
and Cravatt describe how one probe in their library can be used to detect
six or seven mechanistically distinct, known enzyme families.
"This is the first evidence that this technology can be expanded to
cover a significant portion of the proteome," says Cravatt. "The challenge
[now] is to cover the maximum amount."
Sorensen is pursuing projects in total synthesis to produce additional
probes, looking towards bioactive natural products for hints on how to
design them.
"There are a lot of natural products that nature designed to interact
with biological molecules," he says.
One of these, a compound called fumagillin, was discovered in a fungus
that inhibits a virus that infects the bacterium Staphylococcus aureus.
Fumagillin, as it turns out, reacts with certain metalloproteinases and
is a powerful anti-angiogenic and anti-tumor agent.
Sorensen reasoned that the reactivity of the compound might be a good
scaffold upon which to build a more broadly reactive probe. By building
these molecules from commercially available precursors, Sorensen and his
group can design precursors and suitable structural alternatives.
By varying the chemical nature of one of its chains, Sorensen has been
able to redirect fumagillin-like compounds to other proteins in the metalloproteinase
family. Now they are seeing if they can address metalloproteinases in
a global fashion.
Working with Students
Cravatt says projects like these are perfect for students at TSRI because
they offer a twofold education. Students learn both how to make molecules
and test them in living cells to see how they work—a truly interdisciplinary
approach that is rewarding for the students and the instructors alike.
"That's what really drew me—the ability to do both chemistry and
biology," says Adam, who is beginning his fifth year in the Chemistry
Program.
Jessani, who starts his fifth year in the Macromolecular and Cellular
Structure and Chemistry Program this fall, was immediately drawn to the
biological applications of the work. "The serine hydrolases are such a
huge family," he says. "They had developed the probes, and it was obvious
they would be good at looking at biological systems."
"He has really done tremendous interface work," Cravatt adds. And, he
says, TSRI is perfect for investigators like Cravatt and Sorensen who
design such projects because there are students who legitimately want
to work in both fields.
"It's a lot of fun to work with these students," notes Sorensen.
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