Mekdes Debela

Specificity profiling of seven human tissue kallikreins reveals individual subsite preferences.

Human tissue kallikreins (hKs) form a family of 15 closely related (chymo)trypsin-like serine proteinases. These tissue kallikreins are expressed in a wide range of tissues including the central nervous system, the salivary gland, and endocrine-regulated tissues, such as prostate, breast, or testis, and may have diverse physiological functions. For several tissue kallikreins, a clear correlation has been established between expression and different types of cancer. For example, the prostate-specific antigen (PSA or hK3) serves as tumor marker and is used to monitor therapy response. Using a novel strategy, we have cloned, expressed in Escherichia coli or in insect cells, refolded, activated, and purified the seven human tissue kallikreins hK3/PSA, hK4, hK5, hK6, hK7, hK10, and hK11. Moreover, we have determined their extended substrate specificity for the nonprime side using a positional scanning combinatorial library of tetrapeptide substrates. hK3/PSA and hK7 exhibited a chymotrypsin-like specificity preferring large hydrophobic or polar residues at the P1 position. In contrast, hK4, hK5, and less stringent hK6 displayed a trypsin-like specificity with strong preference for P1-Arg, whereas hK10 and hK11 showed an ambivalent specificity, accepting both basic and large aliphatic P1 residues. The extended substrate specificity profiles are in good agreement with known substrate cleavage sites but also in accord with experimentally solved (hK4, hK6, and hK7) or modeled structures. The specificity profiles may lead to a better understanding of human tissue kallikrein functions and assist in identifying their physiological protein substrates as well as in designing more selective inhibitors.

Structures and specificity of the human kallikrein-related peptidases KLK 4, 5, 6, and 7

Abstract Human kallikrein-related peptidases (KLKs) are (chymo)-trypsin-like serine proteinases that are expressed in a variety of tissues such as prostate, ovary, breast, testis, brain, and skin. Although their physiological functions have been only partly elucidated, many of the KLKs appear to be useful prognostic cancer markers, showing distinct correlations between their expression levels and different stages of cancer. Recent advances in the purification of ‘new type’ recombinant KLKs allowed solution of the crystal structures of KLK4, KLK5, KLK6, and KLK7. Along with these data, enzyme kinetic studies and extended substrate specificity profiling have led to an understanding of the non-prime-side substrate preferences of KLK4, 5, 6, and 7. The shape and polarity of the specificity pockets S1–S4 explain well their substrate preferences. KLK4, 5, and 6 exhibit trypsin-like specificity, with a strong preference for Arg at the P1 position of substrates. In contrast, KLK7 displays a unique chymotrypsin-like specificity for Tyr, which is also preferred at P2. All four KLKs show little specificity for P3 residues and have a tendency to accept hydrophobic residues at P4. Interestingly, for KLK4, 5, and 7 extended charged surface regions were observed that most likely serve as exosites for physiological substrates.

Chymotryptic specificity determinants in the 1.0 A structure of the zinc-inhibited human tissue kallikrein 7.

hK7 or human stratum corneum chymotryptic enzyme belongs to the human tissue kallikrein (hKs) serine proteinase family and is strongly expressed in the upper layers of the epidermis. It participates in skin desquamation but is also implicated in diverse skin diseases and is a potential biomarker of ovarian cancer. We have solved x-ray structures of recombinant active hK7 at medium and atomic resolution in the presence of the inhibitors succinyl-Ala-Ala-Pro-Phe-chloromethyl ketone and Ala-Ala-Phe-chloromethyl ketone. The most distinguishing features of hK7 are the short 70–80 loop and the unique S1 pocket, which prefers P1 Tyr residues, as shown by kinetic data. Similar to several other kallikreins, the enzyme activity is inhibited by Zn2+ and Cu2+ at low micromolar concentrations. Biochemical analyses of the mutants H99A and H41F confirm that only the metal-binding site at His99 close to the catalytic triad accounts for the noncompetitive Zn2+ inhibition type. Additionally, hK7 exhibits large positively charged surface patches, representing putative exosites for prime side substrate recognition.

Interplay of human tissue kallikrein 4 (hK4) with the plasminogen activation system: hK4 regulates the structure and functions of the urokinase-type plasminogen activator receptor (uPAR)

Abstract The plasminogen activation system is involved in cancer progression and metastasis. Among other proteolytic factors, it includes the serine protease urokinase-type plasminogen activator (uPA) and its three-domain (D1D2D3) receptor uPAR (CD87), which focuses plasminogen activation to the cell surface. The function of uPAR is regulated in part through shedding of domain D1 by proteases, e.g., uPA itself or plasmin. Human tissue kallikrein 4 (hK4), which is highly expressed in prostate and ovarian tumor tissue, was previously shown to cleave and activate the pro-enzyme forms of prostate-specific antigen (PSA, tissue kallikrein hK3) and uPA. Here we demonstrate that uPAR is also a target for hK4, being cleaved in the D1-D2 linker sequence and, to a lesser extent, in its D3 juxtamembrane domain. hK4 may thus modulate the tumor-associated uPA/uPAR-system activity by either activating the pro-enzyme form of uPA or cleaving the cell surface-associated uPA receptor.

A completed KLK activome profile: investigation of activation profiles of KLK9, 10, and 15

Abstract We previously reported the activation profiles of the human kallikrein-related peptidases (KLKs) as determined from a KLK pro-peptide fusion-protein system. That report described the activity profiles of 12 of the 15 mature KLKs versus the 15 different pro-KLK sequences. The missing profiles in the prior report, involving KLK9, 10, and 15, are now described. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, mass spectrometry, and N-terminal sequence analyses show that KLK9 and 10 exhibit low hydrolytic activities towards all of the 15 pro-KLK sequences, while KLK15 exhibits significant activity towards both Arg- and Lys-containing KLK pro-sequences. The ability of KLK15 to activate pro-KLK8, 12, and 14 is confirmed using recombinant pro-KLK proteins, and shown to be significant for activation of pro-KLK8 and 14, but not 12. These additional data for KLK9, 10, and 15 now permit a completed KLK activome profile, using a KLK pro-peptide fusion-protein system, to be described. The results suggest that KLK15, once activated, can potentially feed back into additional pro-KLK activation pathways. Conversely, KLK9 and 10, once activated, are unlikely to participate in further pro-KLK activation pathways, although similar to KLK1 they may activate other bioactive peptides.

Structure-function analyses of human kallikrein-related peptidase 2 establish the 99-loop as master regulator of activity.

Background: Serine proteases KLK2 and KLK3 clear the way for spermatozoa before impregnation. Results: Enzymatic assays and structures of KLK2 elucidate its catalytic action, especially when compared with conformations of similar proteases. Conclusion: Flexible loops around the active site of serine proteases open concertedly upon substrate binding. Significance: This mechanistic model will stimulate the design of pharmaceutical inhibitors. Human kallikrein-related peptidase 2 (KLK2) is a tryptic serine protease predominantly expressed in prostatic tissue and secreted into prostatic fluid, a major component of seminal fluid. Most likely it activates and complements chymotryptic KLK3 (prostate-specific antigen) in cleaving seminal clotting proteins, resulting in sperm liquefaction. KLK2 belongs to the “classical” KLKs 1–3, which share an extended 99- or kallikrein loop near their non-primed substrate binding site. Here, we report the 1.9 Å crystal structures of two KLK2-small molecule inhibitor complexes. In both structures discontinuous electron density for the 99-loop indicates that this loop is largely disordered. We provide evidence that the 99-loop is responsible for two biochemical peculiarities of KLK2, i.e. reversible inhibition by micromolar Zn2+ concentrations and permanent inactivation by autocatalytic cleavage. Indeed, several 99-loop mutants of KLK2 displayed an altered susceptibility to Zn2+, which located the Zn2+ binding site at the 99-loop/active site interface. In addition, we identified an autolysis site between residues 95e and 95f in the 99-loop, whose elimination prevented the mature enzyme from limited autolysis and irreversible inactivation. An exhaustive comparison of KLK2 with related structures revealed that in the KLK family the 99-, 148-, and 220-loop exist in open and closed conformations, allowing or preventing substrate access, which extends the concept of conformational selection in trypsin-related proteases. Taken together, our novel biochemical and structural data on KLK2 identify its 99-loop as a key player in activity regulation.

Structural basis for the Zn2+ inhibition of the zymogen-like kallikrein-related peptidase 10.

Abstract Although kallikrein-related peptidase 10 (KLK10) is expressed in a variety of human tissues and body fluids, knowledge of its physiological functions is fragmentary. Similarly, the pathophysiology of KLK10 in cancer is not well understood. In some cancer types, a role as tumor suppressor has been suggested, while in others elevated expression is associated with poor patient prognosis. Active human KLK10 exhibits a unique, three residue longer N-terminus with respect to other serine proteases and an extended 99-loop nearly as long as in tissue kallikrein KLK1. Crystal structures of recombinant ligand-free KLK10 and a Zn2+ bound form explain to some extent the mixed trypsin- and chymotrypsin-like substrate specificity. Zn2+-inhibition of KLK10 appears to be based on a unique mechanism, which involves direct binding and blocking of the catalytic triad. Since the disordered N-terminus and several loops adopt a zymogen-like conformation, the active protease conformation is very likely induced by interaction with the substrate, in particular at the S1 subsite and at the unusual Ser193 as part of the oxyanion hole. The KLK10 structures indicate that the N-terminus, the nearby 75-, 148-, and the 99-loops are connected in an allosteric network, which is present in other trypsin-like serine proteases with several variations.

Structural determinants of specificity and regulation of activity in the allosteric loop network of human KLK8/neuropsin

Human KLK8/neuropsin, a kallikrein-related serine peptidase, is mostly expressed in skin and the hippocampus regions of the brain, where it regulates memory formation by synaptic remodeling. Substrate profiles of recombinant KLK8 were analyzed with positional scanning using fluorogenic tetrapeptides and the proteomic PICS approach, which revealed the prime side specificity. Enzyme kinetics with optimized substrates showed stimulation by Ca2+ and inhibition by Zn2+, which are physiological regulators. Crystal structures of KLK8 with a ligand-free active site and with the inhibitor leupeptin explain the subsite specificity and display Ca2+ bound to the 75-loop. The variants D70K and H99A confirmed the antagonistic role of the cation binding sites. Molecular docking and dynamics calculations provided insights in substrate binding and the dual regulation of activity by Ca2+ and Zn2+, which are important in neuron and skin physiology. Both cations participate in the allosteric surface loop network present in related serine proteases. A comparison of the positional scanning data with substrates from brain suggests an adaptive recognition by KLK8, based on the tertiary structures of its targets. These combined findings provide a comprehensive picture of the molecular mechanisms underlying the enzyme activity of KLK8.