KLK5 and KLK7, two members of the human tissue kallikrein family, are differentially expressed in lung cancer

Emerging data indicate that serine proteases of the kallikrein family (KLK) are implicated in various human diseases, including carcinoma; however, kallikrein gene expression has never been investigated in lung cancer. Using RT-PCR and Western blotting, we demonstrated the expression of both KLK5 and KLK7, and their respective proteins (hK5 and hK7) in tumoral and nontumoral lung tissues. Quantitative gene expression was then analyzed in a cohort of 56 patients with non-small cell lung cancer by real-time RT-PCR. KLK5 expression is significantly more expressed in squamous cell carcinoma than in matched nonmalignant lung tissue (P=0.02), whereas expression of KLK7 was decreased in adenocarcinoma (P=0.003). Multivariate analysis revealed diverse correlations between the KLK5 and KLK7 expression levels in nonmalignant and malignant tissues, and clinical parameters, including histotype, metastatic status, and grade. Our findings provide new insight into kallikrein gene expression in hormone-independent carcinoma. Altogether, our results suggest that variability in KLK5 and KLK7 gene expression might be involved in lung tumorigenesis and useful for clinical purposes.     

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

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.     

The putative NTPase Fap7 mediates cytoplasmic 20S pre-rRNA processing through a direct interaction with Rps14

One of the proteins identified as being involved in ribosome biogenesis by high-throughput studies, a putative P-loop-type kinase termed Fap7 (YDL166c), was shown to be required for the conversion of 20S pre-rRNA to 18S rRNA. However, the mechanism underlying this function has remained unclear. Here we demonstrate that Fap7 is strictly required for cleavage of the 20S pre-rRNA at site D in the cytoplasm. Genetic depletion of Fap7 causes accumulation of only the 20S pre-rRNA, which could be detected not only in 43S preribosomes but also in 80S-sized complexes. Fap7 is not a structural component of 43S preribosomes but likely transiently interacts with them by directly binding to Rps14, a ribosomal protein that is found near the 3' end of the 18S rRNA. Consistent with an NTPase activity, conserved residues predicted to be required for nucleoside triphosphate (NTP) hydrolysis are essential for Fap7 function in vivo. We propose that Fap7 mediates cleavage of the 20S pre-rRNA at site D by directly interacting with Rps14 and speculate that it is an enzyme that functions as an NTP-dependent molecular switch in 18S rRNA maturation.     

Mycobacterial PIMs inhibit host inflammatory responses through CD14-dependent and CD14-independent mechanisms

Mycobacteria develop strategies to evade the host immune system. Among them, mycobacterial LAM or PIMs inhibit the expression of pro-inflammatory cytokines by activated macrophages. Here, using synthetic PIM analogues, we analyzed the mode of action of PIM anti-inflammatory effects. Synthetic PIM(1) isomer and PIM(2) mimetic potently inhibit TNF and IL-12 p40 expression induced by TLR2 or TLR4 pathways, but not by TLR9, in murine macrophages. We show inhibition of LPS binding to TLR4/MD2/CD14 expressing HEK cells by PIM(1) and PIM(2) analogues. More specifically, the binding of LPS to CD14 was inhibited by PIM(1) and PIM(2) analogues. CD14 was dispensable for PIM(1) and PIM(2) analogues functional inhibition of TLR2 agonists induced TNF, as shown in CD14-deficient macrophages. The use of rough-LPS, that stimulates TLR4 pathway independently of CD14, allowed to discriminate between CD14-dependent and CD14-independent anti-inflammatory effects of PIMs on LPS-induced macrophage responses. PIM(1) and PIM(2) analogues inhibited LPS-induced TNF release by a CD14-dependent pathway, while IL-12 p40 inhibition was CD14-independent, suggesting that PIMs have multifold inhibitory effects on the TLR4 signalling pathway.     

Human kallikrein-related peptidase 14 (KLK14) is a new activator component of the KLK proteolytic cascade. Possible function in seminal plasma and skin

Human kallikrein-related peptidases (KLKs) are a family of 15 serine proteases mainly known for their biomarker utility in various neoplastic and non-neoplastic diseases. Despite significant progress in understanding their clinical application, little is known about the activation mechanism(s) of this important family of enzymes. Emerging evidence indicates that KLKs are activated in a stepwise manner, which is a characteristic of proteolytic cascades. Thus far, KLK cascades have been implicated in semen liquefaction and skin desquamation. Many members of the KLK family have been reported to be active in seminal plasma and/or skin, suggesting their involvement in common proteolytic cascades. KLK14, in particular, is highly active and has recently been proposed as one of the key trypsin-like proteases involved in skin desquamation. This study aims to elucidate a probable cascade-mediated role of KLK14 by 1) examining KLK14-mediated cleavage of a heptapeptide library encompassing activation sites of the 15 KLKs and 2) verifying activation of certain candidate downstream targets of KLK14 (i.e. pro-KLK1, -KLK3, and -KLK11). Heptapeptides encompassing activation motifs of KLK2, -3, -5, and -11 were cleaved with a high (> or =85%) cleavage efficiency. Activation of these candidates was confirmed using full-length recombinant proteins. Pro-KLK11, -KLK3, and -KLK1 were rapidly activated in a concentration-dependent manner. Pro-KLK3 regulation was bidirectional because activation was followed by inactivation via internal cleavage of active KLK3. We are proposing a putative cascade model, operating through multiple KLKs. Identification of novel members of such proteolytic cascades will aid in further defining mechanisms involved in seminal/skin homeostasis.     

Structure, function and control of complement c5 and its proteolytic fragments

As part of the innate immune system, the complement system recognises a wide range of non-self structures present on pathogens or altered self cells. Its activation elicits proteolytic cascades which eventually results in the cleavage of the C5 protein into two fragments, C5a and C5b. The small anaphylatoxin C5a induces a variety of biological responses upon binding to the 7TM receptors C5aR and the C5L2, while the large C5b fragment nucleates formation of the membrane attack complex capable of killing susceptible pathogens by the formation of a pore structure in association with complement components C6, C7, C8, and C9. A number of regulatory molecules help to control C5 mediated immune responses towards host cells, but in several major inflammatory conditions including sepsis and arthritis, C5a is believed to contribute significantly to disease etiology. Inhibition of membrane attack complex assembly is already approved for treatment of paroxysmal nocturnal haemoglobinuria and atypical hemolytic uremic syndrome. A number of recent crystal structures have provided a comprehensive insight into the architecture and properties of intact C5 and its fragments, and how pathogens interfere with their function. Here we review the functional and structural aspects of C5 and its fragments, the pathological conditions associated with them, and strategies employed by pathogens to interfere with the biological function of C5. Structural insight and elucidation of evasion strategies employed by pathogens present a unique opportunity for promoting the development of novel selective C5 inhibitors with therapeutic applications.     

Inhibition of human kallikreins 5 and 7 by the serine protease inhibitor lympho-epithelial Kazal-type inhibitor (LEKTI)

LEKTI is a 120-kDa protein that plays an important role in skin development, as mutations affecting LEKTI synthesis underlie Netherton syndrome, an inherited skin disorder producing severe scaling. Its primary sequence indicates that the protein consists of 15 domains, all resembling a Kazal-type serine protease inhibitor. LEKTI and two serine proteases belonging to the human tissue kallikrein (hK) family (hK5 and hK7) are expressed in the granular layer of skin. In this study, we characterize the interaction of two recombinant LEKTI fragments containing three or four intact Kazal domains (domains 6-8 and 9-12) with recombinant rhK5, a trypsin-like protease, and recombinant rhK7, a chymotrypsin-like protease. Both fragments inhibited rhK5 similarly in binding and kinetic studies performed at pH 8.0, as well as pH 5.0, the pH of the stratum corneum where both LEKTI and proteases may function. Inhibition equilibrium constants (Ki) measured either directly in concentration-dependent studies or calculated from measured association (kass) and dissociation (kdis) rate constants were 1.2-5.5 nM at pH 8.0 and 10-20 nM at pH 5.0. At pH 8.0, kass and kdis values were 4.7 x 10(5) M(-1) s(-1) and 5.5 x 10(-4) s(-1), and at pH 5.0 they were 4.0 x 10(4) M(-1) s(-1) and 4.3 x 10(-4) s(-1), respectively. The low Ki and kdis values (t1/2 of 20-25 min) indicate tight and specific association. Only fragment 6-9' was a good inhibitor of rhK7, demonstrating a Ki of 11 nM at pH 8.0 in a reaction that was rapidly reversible. These results show that LEKTI, at least in fragment form, is a potent inhibitor of rhK5 and that this protease may be a target of LEKTI in human skin.     

Overexpression of the human tissue kallikrein genes KLK4, 5, 6, and 7 increases the malignant phenotype of ovarian cancer cells

The human tissue kallikrein family of serine proteases (hK1-hK15 encoded by the genes KLK1-KLK15) is involved in several cancer-related processes. Accumulating evidence suggests that certain tissue kallikreins are part of an enzymatic cascade pathway that is activated in ovarian cancer and other malignant diseases. In the present study, OV-MZ-6 ovarian cancer cells were stably co-transfected with plasmids expressing hK4, hK5, hK6, and hK7. These cells displayed similar proliferative capacity as the vector-transfected control cells (which do not express any of the four tissue kallikreins), but showed significantly increased invasive behavior in an in vitro Matrigel invasion assay (p<0.01; Mann-Whitney U-test). For in vivo analysis, the cancer cells were inoculated into the peritoneum of nude mice. Simultaneous expression of hK4, hK5, hK6, and hK7 resulted in a remarkable 92% mean increase in tumor burden compared to the vector-control cell line. Five out of 14 mice in the 'tissue kallikrein overexpressing' group displayed a tumor/situs ratio greater than 0.198, while this weight limit was not exceeded at all in the vector control group consisting of 13 mice (p=0.017; chi2 test). Our results strongly support the view that tumor-associated overexpression of tissue kallikreins contributes to ovarian cancer progression.     

Characterization of SPINK9, a KLK5-specific inhibitor expressed in palmo-plantar epidermis

SPINK9, a Kazal-type serine protease inhibitor, is almost exclusively expressed in the palmo-plantar epidermis. SPINK9 selectively inhibits kallikrein-related peptidase 5 (KLK5), no other target enzyme is known at present. In this study, we defined the reactive loop to residues 48 and 49 of SPINK9 and characterized the inhibition and binding of different SPINK9 variants towards KLK5, KLK7, KLK8 and KLK14. Substitutions of single amino acids in the reactive loop had a large impact on both inhibitory efficiency and specificity. Binding studies showed that it is mainly the dissociation rate that is affected by the amino acid substitutions. The inhibitory effect of wild-type SPINK9 was clearly pH-dependent with an improved effect at a pH similar to that of the outer layers of the skin. Modeling of the enzyme-inhibitor complexes showed that the reactive loop of SPINK9 fits very well into the deep negatively charged binding pocket of KLK5. A decrease in pH protonates His48 of the wild-type protein resulting in a positively charged residue, thereby explaining the observed decreased dissociation rate. Interestingly, substitution with a positively charged amino acid at position 48 resulted in a more efficient inhibitor at higher pH.     

Development of a monoclonal anti-ADAMTS-5 antibody that specifically blocks the interaction with LRP1

The potent aggrecanase ADAMTS-5 is constitutively secreted by chondrocytes, but it is rapidly endocytosed in normal cartilage via the cell surface endocytic receptor LRP1. Therefore it is difficult to detect the total ADAMTS-5 activity produced. In this study, we isolated a monoclonal anti-ADAMTS-5 antibody 1B7 that blocks LRP1-mediated internalization without affecting the aggrecanolytic activity. Addition of 1B7 to cultured human chondrocytes revealed the full aggrecanolytic activity of ADAMTS-5 generated by the cells. 1B7 is a useful tool to estimate the ADAMTS-5 activity and to identify its potential roles in the tissues.     

Anti-innexin 2 aptamers specifically inhibit the heterologous interaction of the innexin 2 and innexin 3 carboxyl-termini in vitro

We recently demonstrated that heteromerization of innexins 2 and 3 from Drosophila melanogaster (Dm) is crucial for epithelial organization and polarity of the embryonic epidermis. Both innexins are thought to interact via their C-terminal cytoplasmic domains during the assembly of heteromeric gap junction channels. However, the mechanisms that control heteromeric versus homomeric channel formation are still largely unknown. Here we report the isolation of both non-modified and 2'-fluoro-2'-deoxy-modified RNA anti-innexin 2 aptamers by in vitro selection. The aptamers bind to a proximal epitope on the carboxyl-tail of Dm innexin 2 protein and specifically inhibit the heterologous interaction of innexin 2 and innexin 3 carboxyl-termini in vitro. These domain-specific inhibitors represent the first step towards functional studies focusing on the activity of these domains in vivo.     

Three yeast proteins that specifically inhibit yeast proteases A, B, and C.

Baker's yeast was found to contain inhibitors of yeast proteases A and C. These two proteins were partially purified, characterized, and compared with the previously described inhibitor of protease B. The A and B inhibitors were very thermostable and were extracted from intact yeast cells at 9k C. The A inhibitor appeared to be a protein with a molecular weight of about 22,000 which could be dissociated into two monomers or chains, both of which had a molecular weight of approximately 11,000. The protease C (carboxypeptidase Y)-inhibitor complex was purified and then partially disociated on an ion-exchange column. The free protease C inhibitor was very unstable, possibly because of destruction by a contaminating protease. Each inhibitor was specific for its corresponding protease and each inhibition was competitive. Whereas proteases A, B, and C destroyed the B inhibitor, only protease B had a pronounced destructive effect on the protease A inhibitor. Pepstatin was found to be a selective inhibitor of protease A, whereas chymostatin and antipain specifically inhibited protease B.     

Non-covalent interaction of 2', 4', 5', 7'-tetrabromo-4, 5, 6, 7-tetrachlorofluorescein with proteins and its application

The interactions of 2', 4', 5', 7'-tetrabromo-4, 5, 6, 7-tetrachlorofluorescein (TBTCF) with BSA, ovalbumin (OVA) and poly-L-lysine (PLYS) at pH 3.70 have been investigated by combination of the spectral correction technique and the Langmuir isothermal adsorption. The active connection actions such as ion pairs, van der Waals' force, hydrogen bond, hydrophobic bond were proposed to explain the non-covalent interaction between TBTCF and BSA, OVA and PLYS. Effects of the electrolyte and high temperature indicated that union of the active connections between TBTCF and BSA and OVA was too firm to be destroyed. The relationship between the binding number of TBTCF and variety fraction of the amino acid residues was analyzed. The binding number of TBTCF depended on the number of positively charged amino acid residues. The other amino acid residues surrounded and seized TBTCF by hydrogen bonds and hydrophobic bonds when the electrostatic attraction pulled TBTCF to link protein. In addition, a novel method named the absorbance ratio difference was established for determination of protein in trace level and was applied with much higher sensitivity than the ordinary method.     

ERalpha and STAT5a cross-talk: interaction through C-terminal portions of the proteins decreases STAT5a phosphorylation, nuclear translocation and DNA-binding

Cross-talk between ERalpha and STAT5a was demonstrated to mediate through a direct physical association between the two proteins. By GST pull-down assays and functional assays with various constructs of ERalpha and STAT5a, it was shown that the C-termini of these two proteins were mainly responsible for this interaction. Furthermore, the interaction between ERalpha and STAT5a was demonstrated to give rise to functional changes in their signaling events. In cell transfection studies, it was shown that ERalpha activation could attenuate PRLR signaling through STAT5a. This ERalpha-mediated attenuation of PRLR signaling was substantiated by observed decreases in the phosphorylation of JAK2 and STAT5a, reduced translocation of STAT5a into the nucleus, and reduced binding of STAT5a onto a GAS-containing nucleotide. Apart from transfected cells, the interaction between ERalpha and STAT5a could also be observed in established breast cancer cell lines of MCF-7 and T-47D in co-immunoprecipitation studies. However, the functional consequence of the interaction in these cancer cells was very different from the transfected HEK293 cells. ER activation could lead to potentiation of PRLR signaling in MCF-7 cells but not in T-47D cells. Conversely, in both MCF-7 and T-47D cells, PRLR activation could lead to attenuation of ER signaling. These data serve to elucidate the mechanisms underlying the ERalpha-STAT5a cross-talk and in demonstrating that the functional consequence of this cross-talk depends on the precise milieus of the intracellular environment.