Structural studies of complexes of kallikrein 4 with wild-type and mutated forms of the Kunitz-type inhibitor BbKI.

Structures of BbKI, a recombinant Kunitz-type serine protease inhibitor from Bauhinia bauhinioides, complexed with human kallikrein 4 (KLK4) were determined at medium-to-high resolution in four crystal forms (space groups P3121, P6522, P21 and P61). Although the fold of the protein was virtually identical in all of the crystals, some significant differences were observed in the conformation of Arg64 of BbKI, the residue that occupies the S1 pocket in KLK4. Whereas this residue exhibited two orientations in the highest resolution structure (P3121), making either a canonical trypsin-like interaction with Asp189 of KLK4 or an alternate interaction, only a single alternate orientation was observed in the other three structures. A neighboring disulfide, Cys191-Cys220, was partially or fully broken in all KLK4 structures. Four variants of BbKI in which Arg64 was replaced by Met, Phe, Ala and Asp were expressed and crystallized, and their structures were determined in complex with KLK4. Structures of the Phe and Met variants complexed with bovine trypsin and of the Phe variant complexed with α-chymotrypsin were also determined. Although the inhibitory potency of these variant forms of BbKI was lowered by up to four orders of magnitude, only small changes were seen in the vicinity of the mutated residues. Therefore, a totality of subtle differences in KLK4-BbKI interactions within the fully extended interface in the structures of these variants might be responsible for the observed effect. Screening of the BbKI variants against a panel of serine proteases revealed an altered pattern of inhibitory specificity, which was shifted towards that of chymotrypsin-like proteases for the hydrophobic Phe and Met P1 substitutions. This work reports the first structures of plant Kunitz inhibitors with S1-family serine proteases other than trypsin, as well as new insights into the specificity of inhibition of medically relevant kallikreins.

Characterization of AMBN I and II Isoforms and Study of Their Ca2+-Binding Properties.

Ameloblastin (Ambn) as an intrinsically disordered protein (IDP) stands for an important role in the formation of enamel-the hardest biomineralized tissue commonly formed in vertebrates. The human ameloblastin (AMBN) is expressed in two isoforms: full-length isoform I (AMBN ISO I) and isoform II (AMBN ISO II), which is about 15 amino acid residues shorter than AMBN ISO I. The significant feature of AMBN-its oligomerization ability-is enabled due to a specific sequence encoded by exon 5 present at the N-terminal part in both known isoforms. In this study, we characterized AMBN ISO I and AMBN ISO II by biochemical and biophysical methods to determine their common features and differences. We confirmed that both AMBN ISO I and AMBN ISO II form oligomers in in vitro conditions. Due to an important role of AMBN in biomineralization, we further addressed the calcium (Ca2+)-binding properties of AMBN ISO I and ISO II. The binding properties of AMBN to Ca2+ may explain the role of AMBN in biomineralization and more generally in Ca2+ homeostasis processes.

Crystal structures of the complex of a kallikrein inhibitor from Bauhinia bauhinioides with trypsin and modeling of kallikrein complexes.

Structures of a recombinant Kunitz-type serine protease inhibitor from Bauhinia bauhinioides (BbKI) complexed with bovine trypsin were determined in two crystal forms. The crystal structure with the L55R mutant of BbKI was determined in space group P64 at 1.94 Šresolution and that with native BbKI in the monoclinic space group P21 at 3.95 Šresolution. The asymmetric unit of the latter crystals contained 44 independent complexes, thus representing one of the largest numbers of independent objects deposited in the Protein Data Bank. Additionally, the structure of the complex with native BbKI was determined at 2.0 Šresolution from P64 crystals isomorphous to those of the mutant. Since BbKI has previously been found to be a potent inhibitor of the trypsin-like plasma kallikrein, it was also tested against several tissue kallikreins. It was found that BbKI is a potent inhibitor of human tissue kallikrein 4 (KLK4) and the chymotrypsin-like human tissue kallikrein 7 (KLK7). Structures of BbKI complexed with the catalytic domain of human plasma kallikrein were modeled, as well as those with KLK4 and KLK7, and the structures were analyzed in order to identify the interactions that are responsible for inhibitory potency.

Profiling system for skin kallikrein proteolysis applied in gene-deficient mouse models.

Kallikrein-related proteases (KLKs) play a critical role in epidermis physiology and have been implicated in skin pathologies such as Netherton syndrome. The contribution of individual KLKs to skin proteolysis is poorly understood. Monitoring of their activities in skin proteome is hampered by overlapping substrate specificities, and there is a need for novel assays. Here, we present a platform of selective and sensitive fluorogenic substrates and inhibitors for profiling KLK5, KLK7 and KLK14. These chemical tools were evaluated using recombinant KLKs and tissue from a unique set of mice deficient in eight combinations of KLKs and their natural regulator LEKTI.

Digestive proteolysis in the Colorado potato beetle, Leptinotarsa decemlineata: Activity-based profiling and imaging of a multipeptidase network.

The Colorado potato beetle (CPB), Leptinotarsa decemlineata, is a major pest of potato plants, and its digestive system is a promising target for development of pest control strategies. This work focuses on functional proteomic analysis of the digestive proteolytic enzymes expressed in the CPB gut. We identified a set of peptidases using imaging with specific activity-based probes and activity profiling with selective substrates and inhibitors. The secreted luminal peptidases were classified as: (i) endopeptidases of cathepsin D, cathepsin L, and trypsin types and (ii) exopeptidases with aminopeptidase (cathepsin H), carboxypeptidase (serine carboxypeptidase, prolyl carboxypeptidase), and carboxydipeptidase (cathepsin B) activities. The proteolytic arsenal also includes non-luminal peptidases with prolyl oligopeptidase and metalloaminopeptidase activities. Our results indicate that the CPB gut employs a multienzyme network of peptidases with complementary specificities to efficiently degrade ingested proteins. This proteolytic system functions in both CPB larvae and adults and is controlled mainly by cysteine and aspartic peptidases and supported by serine and metallopeptidases. The component enzymes identified here are potential targets for inhibitors with tailored specificities that could be engineered into potato plants to confer resistance to CPB.

Complex modulation of peptidolytic activity of cathepsin D by sphingolipids.

Cathepsin D is an aspartic peptidase involved in cellular processes including proliferation and apoptosis and implicated in human pathologies such as cancer and neurodegeneration. Our knowledge about the relationship between proteolysis and bioactive sphingolipids is still very limited. Here, we describe a complex pattern of modulation of the peptidolytic activity of cathepsin D by sphingolipids. A panel of sphingolipid derivatives was screened in a FRET-based assay; these molecules demonstrated negative or positive modulation of cathepsin D peptidolytic activity, depending on the sphingolipid structure. Certain sphingosines and ceramides inhibited cathepsin D in the submicromolar range, and structural requirements for this inhibitory effect were evaluated. The interaction of cathepsin D with sphingolipids was also demonstrated by fluorescence polarization measurements and determined to follow a competitive inhibition mode. In contrast, monoester phosphosphingolipids, especially ceramide-1-phosphate, were identified as activators of cathepsin D peptidolytic activity at submicromolar concentrations. Thus, sphingolipids and phosphosphingolipids, known to be antagonistic in their cell-signaling functions, displayed opposite modulation of cathepsin D. Sphingolipid-based modulators of cathepsin D are potentially involved in the control of cathepsin D-dependent processes and might serve as a scaffold for the development of novel regulators of this therapeutic target.