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Cornell s Samie Jaffrey on Screening for Protease Activity By Cleaved Protein Labeling

Samie Jaffrey
Assistant professor
Department of Pharmacology, Cornell University Weill Medical College

At A Glance

Name: Samie Jaffrey

Position: Assistant professor, Department of Pharmacology, Cornell University Weill Medical College, since 2001.

Background: Postdoc in neuroscience/chemical biology, Johns Hopkins University, 1999-2001.

PhD in neuroscience and MD in medicine, Johns Hopkins University, 1999.

Recently, Samie Jaffrey received a $210,000 grant from the National Institutes of Health to develop a technology for identifying protease targets within proteomes. ProteoMonitor spoke with Jaffrey to find out more about how his technology was developed, and how it will be applied to disease research.

How did you get into working with proteases, and developing a technology to screen for protease activity?

As a postdoc, I studied nitric oxide signaling. Nitric oxide is a chemical which is made in the body. It functions as a neurotransmitter, and it also regulates vascular and blood pressure. The question that I was particularly interested in understanding was how does nitric oxide mediate its effects? It's known that nitric oxide covalently modifies its proteins.

So in 2001, I reported as a postdoc in Dr. Solomon Snyder's lab at Johns Hopkins University one of the first studies of post-translational modifications using proteomic technology.

We were interested in screening the proteome for targets of nitric oxide. And what we did is we developed these chemical techniques to label proteins that were modified with nitric oxide with affinity tags, and we used those affinity to pull out proteins that had this modification, and to specifically identify the complement of proteins in the proteome that can be modified by nitric oxide.

This was important because nitric oxide is a very important drug, and it's an important biological signaling molecule that is important in both health and disease. Understanding its targets is important for understanding ways to therapeutically intervene in diseases and disorders of nitric oxide production.

This was back in 2001, when mass spectrometry hardware and techniques were still rapidly developing.

So with that as a background, I became interested in studying protein modifications and proteins using proteomic technologies. We've been particularly interested in understanding questions that are of biological or medical importance in terms of the functional changes in the proteome during diseases. These functional changes don't simply represent changes in the abundance of proteins, but really how do signaling processes go awry during diseases.

For one of the grants I recently received from the National Institutes of Health, I proposed to develop a technology that we've been pursuing in our laboratory to identify targets for proteases in the body. It turns out that in many diseases, proteases are misregulated — they're turned on or they're turned off, and the misalteration in protease function causes diseases like cancer and rheumatoid arthritis, and plays a role in a variety of neurodegenerative diseases as well.

So the question is — what are the proteins that are being misregulated by these proteases in these diseases? And if we know those protease targets, we can use that information to develop new types of biomarkers and other diagnostic tests for these diseases.

So the question is, 'How can we screen the proteome for targets of protease?' So we've been developing these technologies that allow us to specifically identify proteins that get cleaved. And the way that we do that is we've developed these techniques to label cleaved proteins. And by labeling cleaved proteins, we can subject them to high-throughput mass spectrometry to identify and quantify their levels, and to see how they change in various disease states.

We're hoping to apply that to understand, in particular, a type of protease called caspases. They're very important in cancer and in neurodegenerative diseases, and we're trying to understand what are the targets of those proteases. Using that information, we'll be able to understand these diseases in greater detail, and develop biomarkers and other tools to diagnose these diseases.

How did you develop this technology for labeling cleaved proteins? Was it related to your work with nitric oxide?

Yes, it actually was. It's really a conceptual extension of that. Our work with nitric oxide involved chemically modifying proteins in order to label them in such a way that the modified proteins can be recovered, and modified proteins would exclusively be those modified by nitric oxide.

In the nitric oxide technology, we developed a technique to apply a biotin just on nitric oxide-modified amino acid residues. And by doing this, we could specifically screen the proteome for proteins and peptides that are generated from those proteins that contain biotin. That allowed us to specifically identify proteins that are modified by nitric oxide, and the sites in those proteins that are modified by nitric oxide.

In this technique that we used for studying protease processing, we developed a technique to specifically impart biotin tags on either the N-terminus of proteins or the C-terminus of proteins.

The interesting thing about protease processing is that after a parent protein is cleaved by a protease and produces two daughter proteins, each of those daughter proteins has an N-terminus. One of the daughter proteins has the same N-terminus as the parent. The other daughter proteins has a completely new N-terminus — an N-terminus that was once internal.

So what we do is we profile the N-termini of proteins. When proteases are activated, new N-termini appear because new daughter peptides appear due to the cleavage of parent proteins. By profiling the appearance of new daughter N-termini, we can infer the identity of cleaved protein targets.

By simply taking cell lines that are deficient in specific proteases, or which have proteases overexpressed, we can profile the gain or loss of N-termini of proteins.

The technique that we use to label the N-termini of proteins is very much related to some of the techniques we began to develop when we studied protein modification by nitric oxide.

How do you expect to apply this technology to discovering new biomarkers?

There are certain types of cancer which are associated specifically with the overexpression of certain proteases. For those sorts of cancers that are specifically associated with protease signaling abonormalities, evidence for misregulation of the target would be evidence that these cancers are present. So there are various types of leukemias, in particular, that have abnormalities in protease processing. Those abnormalities in protease processing represent some of the very early steps in the neoplastic process. One way to detect the presence of those cancers would be to determine if abnormal protease processing is occurring. You could specifically screen for specific targets of those proteases, and determine the level of their cleavage.

The mass spectrometry and the proteomics could allow us to identify those targets, but then the screening might be done using an ELISA test, or a Western blot assay, rather than using mass spectrometry.

Was the grant that you received to develop this technology further, or to apply it to disease research?

It's to develop the technique further, and to apply it.

We've got a considerable amount of preliminary data that's allowed us to establish that this technology could work. Now we're applying this technology to tissues, and to real types of cells.

Are there other techniques like this out there?

Not really. I think no one has used the sort of approach that we're using. I think other people are interested in this sort of idea of identifying protease targets, but this is a very technically challenging approach. We're sort of hoping that the way we're approaching it will allow us to identify protease targets in a very high-throughput way, for literally dozens and dozens of proteases. We hope to look at protease processing in many, many different types of diseases.

One thing that we might uncover is that protease processing may be a feature of diseases that were previously unknown to involve these kinds of abnormalities. That also would be very useful for diagnostic criteria and biomarkers.

Do you have any plans to commercialize this technology?

Yes. The technology is already the subject of a provisional patent, and I think the patent will be approved relatively soon.

The long-term goal would be licensing the technology, and making it available to the proteomics community.

I think in general, identifying new roles for abnormal protease processing will be enabled by this technology, as well as finding targets for known proteases and biomarkers for diseases which definitively known to involve proteases.

How long did it take to develop this technology?

I think we've been working on it for two to three years. It's taken a lot to get the preliminary data to establish the feasibility of the technique, which was presented to the NIH.

When do you think the technology will be available for someone else to use?

Probably in about a year or so. We're moving pretty quickly on this.

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