Biomedical Engineering Reference
In-Depth Information
performed to over express DP8- or DP9-green-fluorescent protein (GFP)-
fusion proteins.
136
By this means, DP9 was found to be associated with
impaired cell adhesion, migration, and monolayer wound healing.
136
DP8 was
also associated with impaired migration and wound healing but not cell
adhesion.
136
Both DP8 and DP9 were found to enhance staurosporine strep-
tomyces (STS)-induced apoptosis, while over-expression of DP9 reportedly
increased the rate of spontaneous apoptosis.
136
Interestingly, using enzyme
negative mutants of each protein, the involvement of DP8 and DP9 in these
processes was found to be independent of their enzymatic activity and of the
RGD motif of DP9.
136
Over expression of DP9 was also found to decrease the
mRNA level of proteins known to be associated with cell adhesion; discoidin
domain receptor 1 (DDR1), E-cadherin, and tissue inhibitor of matrix metal-
loproteinases 2 (TIMP-2).
137
Thus, it is possible that the observed phenotypes
arising from DP8- or DP9-over expression may result from direct protein-
protein interactions of these molecules with as-yet unidentified ligands or via
indirect means which alter the expression and function of additional proteins
involved within cell adhesion and migration.
1.5 Protein Structure, Active Site Entry, and
Substrate Specificity
Substrate binding and specificity inherently relies on the conformational
structure of a protease. To date, numerous crystal structures of DP4, often in
complex with substrates or inhibitors, have been resolved along with structures
for FAP
13
and DP6.
18
A number of groups, including our own, have success-
fully expressed and purified recombinant DP8 and DP9, but the crystal
structures of these enzymes are yet to be revealed. Structural information about
DP8 and DP9 is inferred by building homology models using DP4, FAP,
DP6, and PEP structures. In the absence of abundant knowledge regarding
in vivo biological substrates, the substrate specificity of the DPs has been pre-
dominantly derived from in vitro screening of synthetic combinatorial substrate
libraries with purified recombinant protein. Structural aspects relevant to cat-
alytic activity, substrate entry, and substrate specificity of DP4, FAP, DP8, and
DP9 are discussed below.
DP4 is catalytically active as a dimer with each monomer consisting of an a/b
hydrolase domain and an eight-bladed b-propeller domain
16,138
(Figure 1.3).
Crystal-structure resolution has revealed that between these two domains lies a
large cavity of 42nm(
3-4.5) nm, and within this cavity lies a smaller cavity,
forming the catalytic cleft containing the active triad, Ser630, Asp708, and His
740.
15,16
Entry of substrates to the active site located in this large cavity is
hypothesized to occur via two different mechanisms/openings: (1) substrates
may enter/pass through a funnel located in the top opening of the b-propeller
domain or (2) substrates may enter the active site via the much larger, less
restricted, side opening of the b-propeller domain.
15-17,139
In favor of the sec-
ond hypothesis, the entrance via the side opening appears to be the shortest and
B
