 Why activity-based proteome profiling?
In this post-genomic era, profiling approaches are generating a tremendous wealth of information on genomes, transcriptomes, and proteomes of living organisms, describing many different biological processes. The actual activities of proteins, however, are difficult to predict from their presence, as activity is predominantly regulated by various post-translational processes, such as phosphorylation, translocation and processing. Technologies to reveal activities of proteomes will therefore be crucial to uncover an essential layer of information in biological processes. Activity-based proteome profiling (ABPP) is a technique that displays activities of protein classes.  What is Activity-based Proteome Profiling (ABPP)?
For ABPP we use biotinylated (or otherwise labelled) class-specific inhibitors that react with enzymes in an activity-dependent manner. The reaction results in a covalent, irreversible bond between the enzyme and the labelled inhibitor, which enables subsequent analysis under denaturing conditions. Labelled proteins can be purified and detected on protein blots and the identities of these proteins can be determined by mass spectrometry. An example of an activity-based probe is DCG-04, which has been used to display activities of papain-like cysteine proteases in plants.
Which proteins can be targeted?
There are probes described for many different protein classes. Most of these are hydrolytic enzymes, such as proteases. Proteases catalyse the hydrolysis of peptide bonds in proteins and can be classified into different clans, based on their common evolutionary origin (http://www.merops.ac.uk/). There are probes described for papain-like cysteine proteases (clan CA), caspase-like enzymes (clan CD), the proteasome (clan CD) and ubiquitin- and ubiquitin-like proteases (clan CE). There are also photoaffinity probes described for various metallo and aspartic proteases. Beyond the proteases, there are probes described for kinases, phosphatases and glycosidases and various serine hydrolases.
Can you monitor protein activities in living organisms?
Yes, you can. Although the biotinylated probes are usually not membrane-permeable, fluorescent probes are. Another approach that we are taking is a 2-step in vivo labelling procedure, where chemically tagged proteins are extracted and orthogonally biotinylated through ‘click-chemistry’ under controlled conditions.  What to study with ABPP?
ABPP can be used to study many biological proceeses in any organism. We choose to work on plant-pathogen interactions since these are likely to enclose a fascinating molecular battleground where two organisms interfere in each others protein activities. This field of research is large and unexplored, since current research is focussed on understanding plant immunity. The interaction that we focus on is between Pseudomonas syringae pv. tomato and Arabidopsis thaliana because the genomes of both organisms have been sequenced, and this interaction has been intensively studied.
How to study the function of a protein further?
Proteins that show differential activities during the infection of Arabidopsis by Pseudomonas are further investigated to determine the role of the protein in plant-pathogen interactions. We employ various approaches. Following traditional reverse genetics, we test various pathogen assays on Arabidopsis knock-out and overexpression lines and on Nicotiana benthamiana leaves where transcript levels of the endogenous enzymes have been knocked-down by virus-induced gene silencing (VIGS). We also study the function of enzymes through chemical knock-out assays (see targeted chemical genetics).
What is targeted chemical genetics?
To identify the roles of protein activities in biological processes, we are developing targeted chemical genetics. In this approach, we screen probe-derived chemical libraries to make correlations between (1) a phenotype caused by chemical interference of a particular biological process, and (2) the inhibition of an enzyme activity, monitored by in vivo activity-based profiling. This approach is potentially quick and is not hampered by problems from traditional reverse genetic approaches, such as redundancy and lethality. To establish this technology, we are optimising high-throughput phenotyping and in vivo activity-based profiling.
|
