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Scripps Team IDs New Forms of Apoptotic Protease-Kinase Crosstalk Using PROTOMAP System

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Scientists from the Scripps Research Institute have performed a global study of interactions between caspase proteases and phosphorylation in apoptosis, identifying several previously unobserved forms of crosstalk.

In particular, the study, which is detailed in a paper published in the current edition of Cell, found that caspase cleavage can expose new sites for phosphorylation and that phosphorylation, which has typically been thought to protect proteins from cleavage, can, in fact, promote caspase proteolysis.

The findings have potential implications for cancer drug development, and, said Melissa Dix, a Scripps researcher and author on the paper, could lead to the identification of protein biomarkers for monitoring the effectiveness of various chemotherapy strategies.

The research was performed using a modified version of the Protein Topography and Migration Analysis Platform, or PROTOMAP, system, a technology developed several years ago in the lab of Benjamin Cravatt, a Scripps professor and leader of the Cell study.

PROTOMAP combines one-dimensional SDS-PAGE with shotgun proteomics – in this case on a Thermo Fisher Scientific LTQ-Velos Orbitrap – to provide information on both protein molecular weight and sequence, allowing researchers to investigate protein proteolysis.

The platform generates "peptographs" that display the sequence and molecular weight information in a two-dimensional format that allows researchers to "estimate the magnitude of cleavage and also observe the topography of the cleaved proteins," Scripps researcher Gabriel Simon, also an author on the paper, told ProteoMonitor.

Dix, Simon, and Cravatt introduced the original PROTOMAP system in a 2008 Cell paper in which they described how they used it to generate a proteome-wide profile of proteolytic events induced by the intrinsic apoptotic pathway. In their more recent publication, they have built upon the technology, adding a SILAC labeling step to enable quantitation and an immobilized metal-affinity chromatography, or IMAC, step to enrich phosphopeptides.

Addition of these steps was "relatively straightforward," Dix told ProteoMonitor. "We've been doing SILAC techniques in our lab for quite some time … and optimizing the IMAC protocol for peptides coming out of the gel bands was relatively straightforward."

The major changes took place on the informatics side, Simon said, noting that the previous study was "much less sophisticated" because it used spectral counting for quantitation.

"This study is conceptually similar, but it uses all new software tools. It's a combination of established techniques – SILAC, PROTOMAP, IMAC – with a novel set of software tools," he said.

The researchers used the new system to again investigate the intrinsic apoptotic pathway, looking to better understand how caspase protealysis and phosphorylation interact during cell death. Studying Jurkat T-cells, they identified more than 500 apoptosis-specific phosphorylation events, showing they were enriched on cleaved proteins and around sites of caspase cleavage.

They also demonstrated that protein phosphorylation could promote caspase cleavage and that caspase cleavage could expose new sites to phosphorylation – two forms of crosstalk that, Dix said, had not been previously reported.

The researchers had been motivated to look at apoptotic crosstalk between the two mechanisms by a review of caspase-kinase crosstalk published by Duke University researchers Manabu Kurokawa and Sally Kornbluth shortly after the release of the original PROTOMAP paper, Simon said.

The review focused mainly on "phosphorylation protecting substrates from cleavage as well as an extensive literature on phosphorylation of caspases themselves," Simon said. "It motivated us to say, 'Hey, there might be much more extensive crosstalk going on here. How come nobody has looked at this before?'"

"We suspected that it was because it was very technically challenging," he said. "But we thought we had a unique opportunity using the PROTOMAP platform to look at things in a way that they hadn't been looked at before."

The study was meant as "basic research," Simon said. But, he noted, given that many cancer drugs work by apoptosis and by altering kinase signaling, a better understanding of these elements could prove key to developing better cancer therapeutics.

"We think it's likely that phosphorylation networks interact with apoptotic progression in ways that haven't been discovered before," he said. "And so it's likely that by manipulating phosphorylation you might be able to alter the response of cells to apoptotic stimuli. While we weren't looking at that specifically in this study, we think it's a very interesting area for future exploration."

More immediately, Dix said, the researchers are investigating whether any of the cleaved fragments they identified as being phosphorylated in an apoptotic-specific manner could be useful as biomarkers for monitoring the effectiveness of cancer treatment.

If, for instance, such fragments were secreted into the bloodstream, they could potentially be quantified and tracked as a measure of cancer cell death.

Dix said the Scripps team is also expanding its PROTOMAP analysis to the extrinsic apoptotic pathway – which is driven by stimulation of cell receptors — as opposed to the mitochondrial-driven intrinsic pathway. They are also following up on observed interactions between caspase 8 and phosphorylation to more closely investigate how phosphorylation affects that protease's substrate preferences.

Simon noted that while "traditional apoptosis is sort of a proving ground for technologies like [PROTOMAP] … there are many opportunities with many other post-translational modifications like lysine acetylation or ubiquitination [to study] crosstalk between [PTMs] and proteolysis that we think the PROTOMAP technology would really excel at."

Application of the technique to these other modifications would be straightforward, Dix said, essentially requiring just change in the PTM enrichment method.

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