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Molecular Probes and Activity-Based Proteomics
In FAAH, Cravatt has been looking to exploit the molecular
signaling pathways that the body uses when it senses pain
in order to come up with selective targets that can be used
to treat clinical problems. He has been studying basic questions
like how these neurosignaling molecules function in vivo and
what regulates their function.
Another success that the research has spun off is the development
of molecular probes to be used as tools for proteomics.
Proteomics is a new field that attempts to link the genomic
information coming from the human genome initiative to what
is happening inside a cell by looking at which genes are transcribed
in that cell. Then insight into how the genome is expressed
and how it is controlled can be found by, say, comparing the
expression profiles of two different cell types—from
different tissues, organisms, stages of development, or disease
states. Knowing the differential gene expression in healthy
and cancerous cells, for instance, shows which enzymes may
be the major players in that type of cancer.
In general, expression data from cells is revolutionizing
our understanding of how the genome functions in the body.
If the human genome is a one-dimensional map, then the expression
profiles are like adding a second dimension to the map. The
next step is adding another dimension.
"We're trying to create a higher order of proteomics," says
Cravatt, "to develop probes that actually read out changes
in protein activity directly."
Since much of the protein that is expressed in cells undergoes
post-translational modifications, protein–protein interactions,
and other alterations to their activity, not all genes that
are expressed are active as proteins. The blood clotting cascade,
for instance, relies on over a dozen discrete blood factor
enzymes, but these remain in an inactive form in the absence
of the signals received during the cascade.
Many, many more proteins are expressed than are actually
active at any one time. Attached sugars, phosphates, and other
post-translational modifications of these proteins can alter
the expression landscape in the cell. The proteins that are
most important, for instance, may be the ones that are least
expressed.
"All that is invisible to standard proteomic approaches,"
says Cravatt, who is developing chemical probes that "interrogate"
a protein's active site and yield information about whether
it is active or not.
He calls this "active site proteomics."
Active site proteomics relies on first identifying broadly
active compounds that bind to as many as over a hundred members
of an enzyme family. By characterizing the enzymes collectively
rather than individually, a large number of enzymes in a cell
can be profiled with only a few probes. Then the enzymes bound
to each probe can be separated out on gels and identified.
"Our probes will define," says Cravatt, "in a complex proteome
of 300 different members of an enzyme family, whether 10,
20, 30 are active and so on." And the resolution of expression
levels into activities should add depth and scale to the proteome.
One of the principal uses of these probes will be to generate
differential maps of, for instance, a cancerous cell and a
healthy cell. These maps should show the differences in activity
between enzymes in the two types of cells.
"At some level, that is the end game," says Cravatt.
Much of the versatility in Cravatt's research has come as
the result of his attending TSRI's graduate program. Cravatt
came to TSRI in the early 1990s after completing his undergraduate
degree at Stanford University and completed his Ph.D. under
the guidance of Professor Dale Boger, Vice President for Scientific
Affairs and Dean of Graduate Studies Norton Gilula, and President
Richard Lerner.
"That's one of the few decisions in my life that I have
absolutely no regrets about," Cravatt says. "At that point,
there was no other place that was promoting a chemistry–biology
interface. TSRI was preaching something that was totally unique
at that time. I literally did two and a half years of chemistry
and two and a half years of pure biology during my thesis
work."
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