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Research
Peptidases (often referred
to as
proteases)
are enzymes responsible for the cleavage of peptide bonds. They are not
only
involved in digestive processes and protein turnover, but also play a
crucial
role in regulatory processes, such as apoptosis, blood coagulation and
antigen
presentation. It is therefore not surprising that misregulation of
proteases can
lead to a wide variety of diseases.
Our
laboratory is developing small
molecule tools to study proteases,
with a focus on serine proteases and rhomboids
(a class of
intramembrane serine
proteases). With these tools, we study the function of proteases in
biological
processes.
Chemical proteomics
While the number of sequenced
genomes is still increasing, proteomics faces the task to elucidate the
function
of the gene-products, i.e. the
expressed proteins. Most proteomic experiments deal with global
analysis of
protein expression levels in lysates of cells or tissues. However, the
activity
of proteins is not necessarily linked to their abundance. Proteases are
perhaps the best example of this discrepancy, because virtually all
their
members are translated as inactive zymogens, and their activity is
tightly
regulated by post-translational processes. Thus, to uncover the
biological functions
of proteases, it is desirable to monitor the dynamic levels of their
activities.
Figure
1: Schematic
representation of activity-based
proteomics.
Chemical
proteomics (also:
activity-based proteomics) uses small
molecule probes to specifically monitor small subsets of active
enzymes, based
on modification of the active site by certain chemical reactive groups.
In this
way, only active proteases, and not their inactive or inhibitor-bound
counterparts are chemically tagged (see Figure 1). These tags can then
be used
for visualisation, analysis or enrichment of the active protease
species.
Solid phase chemistry of ABPs
One of the major research areas
of our laboratory is the development of new activity-based probes
(ABPs). ABPs
consist of three basic elements (see Figure 2): a reactive warhead that
is able
to modify a protein in an activity-dependent manner, a tag that
facilitates
enrichment and/or visualization, and a spacer that both separates the
two
previous elements and influences the selectivity of the reactive
warhead.
We
make use of solid-phase chemistries
to create straightforward
syntheses that can easily be carried out and facilitate optimisation
for
different protease targets.
Figure
2: The
three elements of an activity-based probe. A tag for detection may
include a radioisotope, a biotin or a fluorophore. The warhead is the
reactive end of the molecule and will determine which class of enzymes
the ABP will target.
Intramembrane proteases
Rhomboids are a recently
discovered family of intra-membrane proteases that use a serine
in their active
site (see Figure 3). They were first discovered in the fruitfly Drosophila melanogaster. Intramembrane
proteases reside within a lipid
bilayer and
cleave specific proteins in their
trans-membrane region. Subsequen tly,
the
cleaved protein is released and can act as an inter- or
intracellular
signal.
Rhomboid
genes are
found
in many diverse organisms. Interestingly, rhomboids from several other
species are also capable of cleaving Drosophila substrates, but
detailed knowledge about their substrate
specificity and functional roles is
lacking.
Unfortunately, the
currently available chemical tools that have
been used to study serine protease
function are incompatible with rhomboids, possibly due to the exclusive
nature
of intra-membrane proteolysis. Our laboratory aims to develop and
validate
small molecules that selectively target rhomboids. With these tools, we
will
study the functional role of rhomboids in bacterial and mammalian
systems.
Figure
3: Schematic
picture of
rhomboid
proteases
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