Interplay between tetrameric structure, enzymatic activity and allosteric regulation of human dipeptidyl-peptidase I.

Human dipeptidyl-peptidase I (DPPI) is a tetrameric enzyme from the family of papain-like cysteine peptidases. It is ubiquitously expressed and plays important roles in general protein turnover, skin homeostasis and proteolytic processing of effector peptidases in immune cells. In this work we investigate allosteric regulation of DPPI and its relation to the oligomeric structure. First, we investigate the functional significance of the tetrameric state by comparing the kinetic properties of the tetrameric form (DPPItet) with a recombinant monomeric form (DPPImono). We find that both forms have very similar kinetic properties for the hydrolysis of a commonly used synthetic substrate. In agreement with previous studies, no cooperativity is observed in the tetramer. The only significant difference between both forms is a higher catalytic rate of DPPImono. We then characterize three compounds, 3'-nitrophthalanilic acid, chlorogenic acid and caffeic acid that affect DPPI activity via kinetic mechanisms consistent with binding outside of the active site. These compounds are the first known modifiers of DPPI that do not act as specific inhibitors. Chlorogenic acid and caffeic acid act as linear mixed and linear catalytic inhibitors, respectively, and do not discriminate between both forms. In contrast, 3'-nitrophthalanilic acid is a hyperbolic inhibitor that binds DPPItet and DPPImono with different affinities and inhibits their activities via different kinetic mechanisms. Altogether, these results show that the tetrameric structure of DPPI is not necessary for enzymatic activity, however, oligomerization-related structural features can play a role in its regulation.

The catalytic domain of cathepsin C (dipeptidyl-peptidase I) alone is a fully functional endoprotease.

Cathepsin C is a tetrameric lysosomal protease that acts as a dipeptidyl-peptidase due to the presence of the exclusion domain that is unique among papain-like cysteine proteases. Here we describe a recombinant form of cathepsin C lacking its exclusion domain (CatCΔEx) produced in a bacterial expression system (E. coli). CatCΔEx is a monomer with endoprotease activity and affinity for hydrophobic residues such as Phe, Leu or Pro, but not Val, in the P2 position. As opposed to cathepsin C, it does not require chloride ions for its activity. Despite lower turnover rates of hydrolysis of synthetic substrates, CatCΔEx has elastolytic and gelatinolytic activity comparable to other cysteine cathepsins.

An allosteric site enables fine-tuning of cathepsin K by diverse effectors.

The cysteine peptidase cathepsin K is a potent collagenolytic enzyme and a promising target for the treatment of osteoporosis. Here, we characterize its allosteric fine-tuning via a recently identified allosteric site. We show that compound NSC94914 binds this site and acts as a specific partial inhibitor of the collagenolytic activity of cathepsin K. We link the functional differences between NSC94914 and known effectors (compound NSC11345 and glycosaminoglycans) to their different modes of interaction with the site. We characterize the allosteric site by site-directed mutagenesis and show that it is involved in specific regulation of the collagenolytic activity of cathepsin K.