(page 2 of 2)

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."

1 | 2 |



Confocal microscopy immunofluorescence images of cerebellar sections of FAAH +/+ (Top) and FAAH -/- (Bottom) models. The green signal is anti-FAAH, and the red signal is propidium iodide, which stains nuclei. The arrowheads in the top panel highlight intense FAAH immunoreactivity in the cell bodies of Purkinje neurons.

















For more information:

The Cravatt Lab