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The DP8 model produced was very similar to the DPIV structure in the hydrolase domain. Analysis of the active site of the DP8 model revealed significant structural conservation in the catalytic triad between DPIV, PEP and ACPH. Further analysis is required to determine whether any differences in the substrate pockets or substrate access tunnel(s) may contribute to DP8’s ability to act as a dipeptidyl peptidase, endopeptidase and acylaminoacyl peptidase. As the structure of fibroblast activation protein has recently been published, an alternative model may be made using this structure together with DPIV to make a model based on two enzymatically active proteins. Simulated docking of substrates and inhibitors into the model may uncover subtle differences between the structures. This may aid in determining the reason for DP8’s multiple enzyme functionality and aid in the improvement of DPIV inhibitor specificity.

We report an expression and purification procedure to produce homogeneous, active human DP8 and DPIV. The most interesting discovery was that DP8 specifically has both prolyl dipeptidyl aminopeptidase and PEP activities. DPIV inhibitors varied in their selectivity against DP8 but all DP8 activities were inhibited by the irreversible DPIV inhibitor ValboroPro. These data indicate that DP8 is a multifunctional enzyme and that therapeutics based on DPIV inhibition should be counter-screened against DP8.

In summary these results strongly support the idea that AAPs and DPIV represent a promising target complex for the pharmacological therapy of T cell-mediated diseases by preserving and enhancing endogenous immunosuppressive mechanisms. Whereas inhibitors of AAPs appear to preferentially act on CD4CD25 regulatory T cells by preserving their immunosuppressive activity via enhanced expression of immunosuppressive cytokines and FOXP3, inhibition of DPIV leads to increased production/release of TGF-β1 and inhibition of cellular proliferation of predominantly activated effector T cells. Thus, specific inhibition of DPIV and AAPs via small molecular compounds provides a new approach for the pharmacological treatment of autoimmune and inflammatory diseases that simultaneously interferes with two major axis of T cell function.

The DP8 model produced was very similar to the DPIV structure in the hydrolase domain. Analysis of the active site of the DP8 model revealed significant structural conservation in the catalytic triad between DPIV, PEP and ACPH. Further analysis is required to determine whether any differences in the substrate pockets or substrate access tunnel(s) may contribute to DP8’s ability to act as a dipeptidyl peptidase, endopeptidase and acylaminoacyl peptidase. As the structure of fibroblast activation protein has recently been published, an alternative model may be made using this structure together with DPIV to make a model based on two enzymatically active proteins. Simulated docking of substrates and inhibitors into the model may uncover subtle differences between the structures. This may aid in determining the reason for DP8’s multiple enzyme functionality and aid in the improvement of DPIV inhibitor specificity.

Potential therapeutic applications of DPIV inhibitors have fuelled interest in understanding the biological roles of DPIV and its relatives. Such efforts are confounded by the ubiquitous expression of DPIV, inhibitor selectivity questions and the variety of identified substrates. DPIV is not essential, but is such a useful enzyme that all animal species express it. The enzyme activity’s ancient and primary function is probably nutritional, providing more complete proteolysis of food and recycled proteins. This function is unnecessary in well-fed humans. The development of selective inhibitors of proteolytic activity and identification of ligand binding activities in this gene family would lead to rapid advances in understanding the biology of the POP gene family.

The DP8 model produced was very similar to the DPIV structure in the hydrolase domain. Analysis of the active site of the DP8 model revealed significant structural conservation in the catalytic triad between DPIV, PEP and ACPH. Further analysis is required to determine whether any differences in the substrate pockets or substrate access tunnel(s) may contribute to DP8’s ability to act as a dipeptidyl peptidase, endopeptidase and acylaminoacyl peptidase. As the structure of fibroblast activation protein has recently been published, an alternative model may be made using this structure together with DPIV to make a model based on two enzymatically active proteins. Simulated docking of substrates and inhibitors into the model may uncover subtle differences between the structures. This may aid in determining the reason for DP8’s multiple enzyme functionality and aid in the improvement of DPIV inhibitor specificity.

The biological significance of the new DPIV family members DP8 and DP9 is unknown. In order to obtain correlations between cell behaviors and peptidase expression levels, DP8 and DP9 overexpression in transfected cells was quantified by expressing green fluorescent protein fusion proteins. We found that, like DPIV and FAP, cells overexpressing DP8 and DP9 exhibit behavioral changes in the presence of ECM components. We demonstrated that these effects were independent of enzyme activity, and of the RGD motif that occurs in DP9. This study is the first indication of some similarities as well as differences between DP8, DP9, DPIV and FAP in their cell biological roles.

In summary these results strongly support the idea that AAPs and DPIV represent a promising target complex for the pharmacological therapy of T cell-mediated diseases by preserving and enhancing endogenous immunosuppressive mechanisms. Whereas inhibitors of AAPs appear to preferentially act on CD4CD25 regulatory T cells by preserving their immunosuppressive activity via enhanced expression of immunosuppressive cytokines and FOXP3, inhibition of DPIV leads to increased production/release of TGF-β1 and inhibition of cellular proliferation of predominantly activated effector T cells. Thus, specific inhibition of DPIV and AAPs via small molecular compounds provides a new approach for the pharmacological treatment of autoimmune and inflammatory diseases that simultaneously interferes with two major axis of T cell function.

Endothelia cells in sparse culture are migratory and increase the production of gelatinases of serine- and metallo-classes in membrane vesicles. Collectively, proteases associated with membrane vesicles degrade extracellular matrix components including type-I and type-IV collagens, laminin and fibronectin. Inhibitor studies suggest the existence of small gelatinases that were derived from these serine- and metallo-proteases. Thus, further studies are warranted to demonstrate the cooperative action of metallo- and serine proteases on cell surfaces and in extracellular vesicles during endothelial cell migration in 3D collagenous matrices, and potential proteolytic activation mechanism for these cell surface proteases.

We report an expression and purification procedure to produce homogeneous, active human DP8 and DPIV. The most interesting discovery was that DP8 specifically has both prolyl dipeptidyl aminopeptidase and PEP activities. DPIV inhibitors varied in their selectivity against DP8 but all DP8 activities were inhibited by the irreversible DPIV inhibitor ValboroPro. These data indicate that DP8 is a multifunctional enzyme and that therapeutics based on DPIV inhibition should be counter-screened against DP8.