Bojan Doljak

Screening of selected food and medicinal plant extracts for pancreatic lipase inhibition

Lipids are important components in human nutrition; however, their increased intake contributes to the development of obesity and can lead to multiple long‐term complications. Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is a key enzyme for the absorption of dietary triglycerides. Interference with fat hydrolysis results in the reduced utilization of ingested lipids, therefore inhibition of lipases decreases fat absorption. Extracts from 106 species of medicinal plants, vegetables and fruits were screened for potential lipase inhibitory activity. p‐Nitrophenylpalmitate and 5‐bromo‐4‐chloro‐3‐indoxylpalmitate were used as substrates in an in vitro test with crude porcine pancreatic lipase. Bearberry (Arctostaphylos uva‐ursi), garden pea (Pisum sativum), Norway spruce (Picea abies) and large‐leaved lime (Tilia platyphyllos) extracts were the most active. Additionally, the activity of selected extracts with removed polyphenols was measured. Extracts of bearberry, garden pea and large‐leaved lime are a promising source for developing functional foods or isolating active compounds. Copyright

Role of Cysteine Cathepsins in Matrix Degradation and Cell Signalling

Cysteine cathepsins participate in extracellular matrix (ECM) degradation and remodelling and thus influence important cellular processes such as cell transformation and differentiation, motility, adhesion, invasion, angiogenesis, and metastasis. Also, cathepsins are involved in cell signalling and are capable of activating specific cell receptors and growth factors or liberating them from the ECM. In this review we emphasize recent studies on cathepsins in regard to ECM degradation and cell signalling.

Cathepsin X cleaves the C-terminal dipeptide of alpha- and gamma-enolase and impairs survival and neuritogenesis of neuronal cells

The cysteine carboxypeptidase cathepsin X has been recognized as an important player in degenerative processes during normal aging and in pathological conditions. In this study we identify isozymes alpha- and gamma-enolases as targets for cathepsin X. Cathepsin X sequentially cleaves C-terminal amino acids of both isozymes, abolishing their neurotrophic activity. Neuronal cell survival and neuritogenesis are, in this way, regulated, as shown on pheochromocytoma cell line PC12. Inhibition of cathepsin X activity increases generation of plasmin, essential for neuronal differentiation and changes the length distribution of neurites, especially in the early phase of neurite outgrowth. Moreover, cathepsin X inhibition increases neuronal survival and reduces serum deprivation induced apoptosis, particularly in the absence of nerve growth factor. On the other hand, the proliferation of cells is decreased, indicating induction of differentiation. Our study reveals enolase isozymes as crucial neurotrophic factors that are regulated by the proteolytic activity of cathepsin X.

Cytokeratin 8 ectoplasmic domain binds urokinase-type plasminogen activator to breast tumor cells and modulates their adhesion, growth and invasiveness

BackgroundGeneration of plasmin is a characteristic of tumor cells, promoting the degradation of extracellular matrix, tumor progression and metastasis. The process is accelerated if plasminogen and plasminogen activator are bound to their cell surface receptors.ResultsIn this study we show that the monoclonal antibody that recognizes an epitope on the cytokeratin 8 (CK8) ectoplasmic domain (anti-CK MAb) inhibits plasminogen activation mediated by urokinase-type plasminogen activator (uPA) in MCF-7 and MCF-10A neoT cells. The ectoplasmic domain of CK8 acts as a binding site for plasminogen, however, by using confocal microscopy, we demonstrated that it is also co-localized with uPA. CK8, therefore, function also as a receptor for uPA on the cell surface, and the presence of anti-CK MAb may prevent the binding of uPA to a designated CK8 motif. The consequent inhibition of plasmin generation resulted in changed cell morphology, enhanced cell adhesion to fibronectin, reduced invasion potential, and an enhanced G1/S transition. Moreover, surface plasmon resonance analysis showed that the synthetic dodecapeptide corresponding to the epitope sequence (VKIALEVEIATY), binds uPA in the nanomolar range.ConclusionThese novel findings suggest a model in which CK8, together with uPA, plasminogen and fibronectin, constitutes a signaling platform capable of modulating cell adhesion/growth-dependent signal transduction in breast tumor cells. Anti-CK MAb, which competes for the binding site for uPA, could be used as an agent to reduce the invasive potential of breast tumor cells.

Cysteine cathepsins: regulators of antitumour immune response

Cysteine cathepsins are lysosomal cysteine proteases that are involved in a number of important biological processes, including intracellular protein turnover, propeptide and hormone processing, apoptosis, bone remodelling and reproduction. In cancer, the cathepsins have been linked to extracellular matrix remodelling and to the promotion of tumour cell motility, invasion, angiogenesis and metastasis, resulting in poor outcome of cancer patients; however, cysteine cathepsins are also involved at different levels of the innate and adaptive immune responses. Their best known role in this aspect is their contribution to major histocompatibility complex class II antigen presentation, the processing of progranzymes into proteolytically active forms, cytotoxic lymphocyte self-protection, cytokine and growth factor degradation and, finally, the induction of cytokine expression and modulation of integrin function. This review is focused on the role of cysteine cathepsins in the antitumour immune response and the evaluation of their pro- and anticancer behaviours during the regulation of these processes.

Cathepsin H Mediates the Processing of Talin and Regulates Migration of Prostate Cancer Cells

Background: Cathepsin H (CtsH) is an aminopeptidase that is involved in tumor progression. Results: CtsH cleaves talin, and its inhibition reduces the migration of prostate cancer cells. Conclusion: CtsH affects cell migration by influencing the activity of integrins, a process that could be regulated by talin cleavage. Significance: Identification of novel CtsH proteolytic targets is important to understand and control tumor progression. The cytoskeletal protein talin, an actin- and β-integrin tail-binding protein, plays an important role in cell migration by promoting integrin activation and focal adhesion formation. Here, we show that talin is a substrate for cathepsin H (CtsH), a lysosomal cysteine protease with a strong aminopeptidase activity. Purified active CtsH sequentially cleaved a synthetic peptide representing the N terminus of the talin F0 head domain. The processing of talin by CtsH was determined also in the metastatic PC-3 prostate cancer cell line, which exhibits increased expression of CtsH. The attenuation of CtsH aminopeptidase activity by a specific inhibitor or siRNA-mediated silencing significantly reduced the migration of PC-3 cells on fibronectin and invasion through Matrigel. We found that in migrating PC-3 cells, CtsH was co-localized with talin in the focal adhesions. Furthermore, specific inhibition of CtsH increased the activation of αvβ3-integrin on PC-3 cells. We propose that CtsH-mediated processing of talin might promote cancer cell progression by affecting integrin activation and adhesion strength.

Comparison of bacterial and phage display peptide libraries in search of target-binding motif

Genetic engineering allows modification of bacterial and bacteriophage genes, which code for surface proteins, enabling display of random peptides on the surface of these microbial vectors. Biologic peptide libraries thus formed are used for high-throughput screening of clones bearing peptides with high affinity for target proteins. There are reports of many successful affinity selections performed with phage display libraries and substantially fewer cases describing the use of bacterial display systems. In theory, bacterial display has some advantages over phage display, but the two systems have never been experimentally compared. We tested both techniques in selecting streptavidin-binding peptides from two commercially available libraries. Under similar conditions, selection of phage-displayed peptides to model protein streptavidin proved convincingly better.

Inactivation of harmful tumour-associated proteolysis by nanoparticulate system

The primary aim in cancer therapy is to deliver anti-cancer drugs to their specific molecular targets in the tumour. Here we present a system composed of poly(d,l-lactide-co-glycolide) nanoparticles, cytokeratin specific monoclonal antibody and cystatin, a potent protease inhibitor, that can neutralize the excessive proteolytic activity associated with the invasive and metastatic potential of breast tumour cells. The antibody provides specific targeting of the delivery system to invasive breast epithelial cells and, additionally, prevents the generation of plasmin, a central extracellular protease involved in malignant progression. Polymeric nanoparticles rapidly enter the targeted cells and release the inhibitor cargo within the endosomes/lysosomes. The inhibitor is capable to inactivate lysosomal cysteine proteases, in particular cathepsin B, which is involved in the degradation of extracellular matrix inside the tumour cells. Our approach, which combines nanoparticulate delivery system with the inhibitory potential against extracellular and intracellular proteases, may improve the efficacy of therapy in patients with breast tumours compared to the application of individual protease inhibitors.

Profilin 1 as a target for cathepsin X activity in tumor cells.

Cathepsin X has been reported to be a tumor promotion factor in various types of cancer; however, the molecular mechanisms linking its activity with malignant processes are not understood. Here we present profilin 1, a known tumor suppressor, as a target for cathepsin X carboxypeptidase activity in prostate cancer PC-3 cells. Profilin 1 co-localizes strongly with cathepsin X intracellularly in the perinuclear area as well as at the plasma membrane. Selective cleavage of C-terminal amino acids was demonstrated on a synthetic octapeptide representing the profilin C-terminal region, and on recombinant profilin 1. Further, intact profilin 1 binds its poly-L-proline ligand clathrin significantly better than it does the truncated one, as shown using cathepsin X specific inhibitor AMS-36 and immunoprecipitation of the profilin 1/clathrin complex. Moreover, the polymerization of actin, which depends also on the binding of poly-L-proline ligands to profilin 1, was promoted by AMS-36 treatment of cells and by siRNA cathepsin X silencing. Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X. The latter is thus designated as a target for development of new antitumor strategies.

Cathepsin X cleavage of the β2 integrin regulates talin‐binding and LFA‐1 affinity in T cells

T cell migration, essential for immune surveillance and response, is mediated by the integrin LFA‐1. CatX, a cysteine carboxypeptidase, is involved in the regulation of T cell migration by interaction with LFA‐1. We show that sequential cleavage of C‐terminal amino acids from the β2 cytoplasmic tail of LFA‐1, by CatX, enhances binding of the adaptor protein talin to LFA‐1 and triggers formation of the latterˈs high‐affinity form. As shown by SPR analysis of peptides constituting the truncated β2 tail, the cleavage of three C‐terminal amino acids by CatX resulted in a 1.6‐fold increase of talin binding. Removal of one more amino acid resulted in a 2.5‐fold increase over the intact tail. CatX cleavage increased talin‐binding affinity to the MD but not the MP talin‐binding site on the β2 tail. This was shown by molecular modeling of the β2 tail/talin F3 complex to be a result of conformational changes affecting primarily the distal‐binding site. Analysis of LFA‐1 by conformation‐specific mAb showed that CatX modulates LFA‐1 affinity, promoting formation of high‐affinity from intermediate‐affinity LFA‐1 but not the initial activation of LFA‐1 from a bent to extended form. CatX post‐translational modifications may thus represent a mechanism of LFA‐1 fine‐tuning that enables the trafficking of T cells.