New Protein Survey Upends Understanding of Cell Death Process

By Mark Schrope

Results from a Scripps Research Institute study have nearly tripled the number of proteins known to be involved in the critical process of programmed cell death, and have shot down a long-held idea about the life cycle of proteins. The work opens doors for potential new drug discoveries, and may open countless more as the new technique that enabled the work, dubbed the PROTOMAP, is applied to studies of other cellular functions.

The study was published in the August 22 issue of the journal Cell.

The focus of the research was proteolysis. This is the simple act of enzymes breaking down larger proteins into smaller components, though the impact of this process is far from simple. More than 500 human enzymes are predicted to be involved in proteolysis, a critical driver in processes including blood coagulation; the onset of cancer and infectious diseases; and apoptosis, or programmed cell death, which is tied to conditions such as Huntington's disease and stroke.  

Despite the importance of proteolysis, understanding of the process has remained surprisingly incomplete. This is due in part to limitations of past methods, which could not provide complete information about either the proteins involved, or the products resulting from their breakdown. Benjamin Cravatt, chair of the Chemical Physiology Department at Scripps Research, and his team wanted to begin filling in the many gaps in the understanding of proteolysis, including the full range of proteins broken down during specific cellular processes and what happens to the resulting protein fragments.

The method the scientists developed, which they named PROtein Topography and Migration Analysis Platform (PROTOMAP), combines cutting-edge technologies in a new and powerful way.

The researchers chose apoptosis as the focus of their initial efforts, because substantial information is available about programmed cell death and the proteins involved, to which the researchers could compare their results.

Cravatt and his colleagues processed samples of cells undergoing apoptosis, as well as control samples of intact cells, using electrophoresis. This technique relies on electric currents to separate biological components, which migrate differing distances in a gel according to their size. The scientists then sliced gels at set intervals and used mass spectrometry to identify the proteins found in each slice.

A key aspect of PROTOMAP is a new bioinformatics tool the group devised that analyzes mass spectroscopy data and presents it in a way that is readily interpretable and searchable. PROTOMAP enabled the scientists to access broad views of the entire landscape of proteins in the cells studied and to discern patterns that were completely unexpected.

Rethinking the Life of a Protein

Using PROTOMAP, the Cravatt group characterized 91 proteins known to play roles in apoptosis, but they also found a whopping 170 additional proteins not previously known to be cleaved during apoptosis, nearly tripling the number of known players. Even more surprising was what the team learned about how those proteins may function.

The prevailing thought has been that protein breakdown during proteolysis is in most cases a stepwise process of disassembling proteins for disposal, with only a few of the breakdown products remaining to serve specific functions. The team's findings point to a different perspective. The results suggest that the vast bulk of protein breakdown products actually serve new functions, with only a few being fully degraded.

"The fact that only a handful disappeared was amazing to me," says Cravatt.

The bold conclusions—that past assumptions of the purpose of proteolysis in apoptosis may need to be revised and that breakdown products continue to lead productive lives—are supported by two lines of argument. First, many of the breakdown products revealed sequences that match known, functional protein domains. Second, these products clearly persisted for extended periods of time in the cells undergoing apoptosis; if proteolysis were merely a disposal mechanism, further breakdown to amino acids—the initial building blocks of proteins—would be expected.

Cravatt says the many new proteins the work has identified should be studied more closely, in part because some may prove good targets for drugs to treat conditions involving apoptosis gone awry, such as strokes, during which brain cells are killed prematurely. Cravatt is also hoping to explore other cellular functions where proteolysis plays a critical role. He says the success in finding so much new information about apoptosis, a process thought to be well understood, bodes well for additional studies.

"That emboldens me to believe that when we go to less understood processes," says Cravatt, "we're going to find even more information."

The Cravatt team also plans to use PROTOMAP techniques to map the proteins and enzymes involved in various forms of cancer.

Other authors on the study, "Global Mapping of the Topography and Magnitude of Proteolytic Events in Apoptosis," were Melissa Dix and Gabriel Simon, both graduate students of The Kellogg School of Science at Technology at Scripps Research. See http://www.cell.com/content/article/abstract?uid=PIIS0092867408008258.

The work was supported through the National Institutes of Health, the ARCS Foundation, a Koshland Graduate Fellowship in Enzyme Biochemistry, and the Skaggs Institute for Chemical Biology.

Send comments to: mikaono[at]scripps.edu

"That emboldens me to believe that when we go to less understood processes, we're going to find even more information."

—Ben Cravatt