Zala Jevnikar

University of Ljubljana, Ljubljana, Ljubljana, Slovenia

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Publications (23)75.43 Total impact

  • Ursa Pecar Fonovic, Zala Jevnikar, Janko Kos
    Biological Chemistry 10/2013; 394(10):1349-52. · 2.96 Impact Factor
  • Urša Pečar Fonović, Zala Jevnikar
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    ABSTRACT: Abstract CX3CL1 chemokine (fractalkine) is highly expressed by vascular smooth muscle cells (VSMC) in atherosclerotic lesions. Its membrane bound form promotes cell-cell interactions while the soluble form induces chemotaxis of CX3CR1- expressing leukocytes. We show that cysteine protease cathepsin S, expressed by VSMC, is able to cleave membrane anchored CX3CL1, releasing a 55kDa fragment to the medium and thus regulates the adhesion of VSMC and the capture of monocytes to the sites of atherogenesis. Moreover, strong co-localization of cathepsin S and CX3CL1 with a recycling endosome marker Rab11a suggests a processing of CX3CL1 in recycling endosomes during its redistribution to the plasma membrane.
    Biological Chemistry 07/2013; · 2.68 Impact Factor
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    ABSTRACT: 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.
    PLoS ONE 01/2013; 8(1):e53918. · 3.53 Impact Factor
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    ABSTRACT: 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 showed 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-terminal of talin F0 head domain. The processing of talin by CtsH was determined also in the metastatic PC-3 prostate cancer cell line, exhibiting increased expression of CtsH. The attenuation of CtsH aminopeptidase activity by specific inhibitor or siRNA-mediated silencing significantly reduced the migration of PC-3 cells on fibronectin, as well as the invasion through Matrigel. We found that in migrating PC-3 cells CtsH was co-localized with talin in the focal adhesions. Further, 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.
    Journal of Biological Chemistry 11/2012; · 4.65 Impact Factor
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    ABSTRACT: Podosomes, specialized actin-rich structures in macrophages, degrade the extracellular matrix (ECM) and are involved in cell migration. On two-dimensional surfaces macrophages form spot-like podosomes at the ventral cell surface that develop into protrusive structures in a three-dimensional (3D) environment resembling the ECM. We have shown that the tips of these protrusive podosomes are characterized by increased accumulation of cysteine cathepsins (Cts) B, X, S, H and L, both in human blood macrophages and in human monocytic cell line U-937. Monocyte-to-macrophage differentiation induces an increase in cysteine cathepsin expression and activity, promoting their translocation to the cell surface, where they interact with ECM. This group of proteases is crucial for the extracellular as well as intracellular degradation of ECM, as demonstrated by quantitative monitoring of collagen IV degradation. Furthermore, inhibiting CtsB, X and S significantly impairs macrophage invasion through the 3D matrix. Time-lapse live-cell imaging of CtsB activity revealed that the extracellular and the intracellular ECM degradation are associated with extensive endocytosis at the tip of protrusive podosomes. The targeting of cysteine cathepsins, as the major mediators of human macrophage 3D invasion, could be an approach to the treatment of inflammatory and cancerous diseases.
    European Journal of Immunology 09/2012; · 4.97 Impact Factor
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    ABSTRACT: Cathepsin H is a unique member of the cysteine cathepsins that acts primarily as an aminopeptidase. Like other cysteine cathepsins, it is synthesized as an inactive precursor and activated by proteolytic removal of its propeptide. Here we demonstrate that, in human cells, the processing of the propeptide is an autocatalytic, multistep process proceeding from an inactive 41kDa pro-form, through a 30kDa intermediate form, to the 28kDa mature form. Tyr87P and Gly90P were identified as the two major endopeptidase cleavage sites, converting the 30kDa form into the mature 28kDa form. The level of processing differs significantly in different human cell lines. In monocyte-derived macrophages U937 and prostate cancer cells PC-3, the 28kDa form is predominant, whereas in osteoblasts HOS the processing from the 30kDa form to the 28kDa form is significantly lower. The aminopeptidase activity of the enzyme and its subcellular localization are independent of the product, however the 30kDa form was not secreted in HOS cells. The activity of the resulting cathepsin H in U937 cells was significantly lower than that in HOS cells, presumably due to the high levels of endogenous cysteine protease inhibitor cystatin F present specifically in this cell line. These results provide an insight into the dependence of human cathepsin H processing and regulation on cell type.
    European journal of cell biology 06/2012; 91(10):757-64. · 3.31 Impact Factor
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    ABSTRACT: Cathepsin H is a cysteine protease considered to play a major role in tumor progression, however, its precise function in tumorigenesis is unclear. Cathepsin H was recently proposed to be involved in processing of bone morphogenetic protein 4 (BMP-4) in mice. In order to clarify whether cathepsin H also regulates BMP-4 in humans, its impact on BMP-4 expression, processing and degradation was investigated in prostate cancer (PC-3), osteosarcoma (HOS) and pro-monocytic (U937) human cell lines. BMP-4 expression was founded to be regulated by cathepsin H using PCR array technology and confirmed by real time PCR. Immunoassays including Western blot and confocal microscopy were used to evaluate the influence of cathepsin H on BMP-4 processing. In contrast to HOS, the expression of BMP-4 mRNA in U937 and PC3 cells was significantly decreased by cathepsin H. The different regulation of BMP-4 synthesis could be associated with the absence of the mature 28 kDa cathepsin H form in HOS cells, where only the intermediate 30 kDa form was observed. No co-localization of BMP-4 and cathepsin H was observed in human cell lines and the multistep processing of BMP-4 was not altered in the presence of specific cathepsin H inhibitor. Isolated cathepsin H does not cleave mature recombinant BMP-4, neither with its amino- nor its endopeptidase activity. Our results exclude direct proteolytic processing of BMP-4 by cathepsin H, however, they provide support for its involvement in the regulation of BMP-4 expression.
    Radiology and Oncology 12/2011; 45(4):259-66. · 1.60 Impact Factor
  • Z Jevnikar, J Kos
    Atlas of Genetics and Cytogenetics in Oncology and Haematology. 11/2011;
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    Zala Jevnikar, Nataša Obermajer, Janko Kos
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    ABSTRACT: The adhesion molecule lymphocyte function-associated antigen (LFA)-1 plays a key role in immune surveillance and response. Its conformation is spatially and temporally regulated, enabling adhesion and deadhesion during T-cell migration. LFA-1 adhesion to its major ligand intercellular adhesion molecule 1 is controlled by adaptor proteins which bind the cytoplasmic tail of the β (2) subunit. Cathepsin X, a cysteine carboxypeptidase, promotes T-cell migration and morphological changes by cleaving the β (2) cytoplasmic tail of LFA-1. In this way, it modulates the affinity of LFA-1 for structural adaptors talin-1 and α-actinin-1 and enables the stepwise transition between intermediate and high-affinity conformations of LFA-1, an event that is necessary for effective T-cell function. Cathepsin X regulation that would allow precise modulation of LFA-1 affinity has a great potential for anti-LFA-1 therapy.
    International Union of Biochemistry and Molecular Biology Life 07/2011; 63(9):686-93. · 2.79 Impact Factor
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    ChemMedChem 05/2011; 6(8):1351-6. · 2.84 Impact Factor
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    ABSTRACT: 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 β(2) cytoplasmic tail of LFA-1, by CatX, enhances binding of the adaptor protein talin to LFA-1 and triggers formation of the latter's high-affinity form. As shown by SPR analysis of peptides constituting the truncated β(2) 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 β(2) tail. This was shown by molecular modeling of the β(2) 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.
    Journal of leukocyte biology 03/2011; 90(1):99-109. · 4.99 Impact Factor
  • Z Jevnikar, J Kos
    02/2011;
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    ABSTRACT: Lactococcus lactis is a lactic acid bacterium of proven safety for use in human oral applications. For this purpose, surface display of recombinant proteins is important, and new approaches for it are being sought. Analysis of the bacterial surface proteome is essential in identifying new candidate carrier proteins for surface display. We have made two different predictions of surface-associated proteins of L. lactis MG1363 by using Augur and LocateP software, which yielded 666 and 648 proteins, respectively. Surface proteins of L. lactis NZ9000, a derivative of MG1363, were identified by using a proteomics approach. The surface proteins were cleaved from intact bacteria, and the resulting peptides were identified by mass spectrometry. The latter approach yielded 80 proteins, 34 of which were not predicted by either software. Of the 80 proteins, 7 were selected for further study. These were cloned in frame with a C-terminal hexahistidine tag and overexpressed in L. lactis NZ9000 using nisin-controlled expression. Proteins of correct molecular weight carrying a hexahistidine tag were detected. Their surface localization was confirmed with flow cytometry. Basic membrane protein A (BmpA) was exposed at the highest level. To test BmpA as a candidate carrier protein, the hexahistidine tag was replaced by the B domain of staphylococcal protein A in the genetic construct. The B domain was displayed on the surface with BmpA as a carrier. The advantage of covalent BmpA binding was demonstrated. BmpA was thus shown to be a suitable candidate for a carrier protein in lactococcal surface display.
    Applied and Environmental Microbiology 02/2011; 77(4):1292-300. · 3.95 Impact Factor
  • International Journal of Medicinal Mushrooms 01/2010; 12(3):245-255. · 0.84 Impact Factor
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    ABSTRACT: The motility of T cells depends on the dynamic spatial regulation of integrin-mediated adhesion and de-adhesion. Cathepsin X, a cysteine protease, has been shown to regulate T-cell migration by interaction with lymphocyte function associated antigen-1 (LFA-1). LFA-1 adhesion to the ICAM-1 is controlled by the association of actin-binding proteins with the cytoplasmic tail of the beta(2) chain of LFA-1. Cleavage by cathepsin X of the amino acid residues S(769), E(768) and A(767) from the C-terminal of the beta(2) cytoplasmic tail of LFA-1 is shown to promote binding of the actin-binding protein alpha-actinin-1. Furthermore, cathepsin X overexpression reduced LFA-1 clustering and induced an intermediate affinity LFA-1 conformation that is known to associate with alpha-actinin-1. Increased levels of intermediate affinity LFA-1 resulted in augmented cell spreading due to reduced attachment of T cells to the ICAM-1-coated surface. Gradual cleavage of LFA-1 by cathepsin X enables the transition between intermediate and high affinity LFA-1, an event that is crucial for effective T-cell migration.
    European Journal of Immunology 09/2009; 39(11):3217-27. · 4.97 Impact Factor
  • Zala Jevnikar, Natasa Obermajer, Janko Kos
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    ABSTRACT: T cells migrate through restrictive barriers in a protease-independent, amoeboid fashion that is characterized by morphological cell polarization. The interaction of cysteine-dependent carboxypeptidase cathepsin X with beta(2) integrin LFA-1 (lymphocyte function associated antigen 1) induces T-cell morphological changes, displaying into a 3D extracellular matrix a cytoplasmic projection termed a uropod. In the present study we show that inhibition of cathepsin X and a cysteine-dependent endopeptidase, cathepsin L, markedly inhibits T-cell actin polymerization, shape polarization, and chemotaxis. We propose that cathepsin L promotes T-cell migration associated processes by activating procathepsin X in the endolysosomal vesicles near the cell membrane and at the peak of the uropod, where both proteases were colocalized. We show that active cathepsin X modifies the beta(2) cytoplasmic tail of LFA-1 in the uropod, promoting its high affinity conformation. We suggest that LFA-1 cleavage contributes to the conformational change in the cytoplasmic tail, promoting the binding of the cytoskeletal protein talin. This interaction is restricted to the uropod and results in the stabilization of this region, promoting LFA-1-mediated cell uropod elongation.
    Cell Motility and the Cytoskeleton 09/2009; 66(11):1030-40. · 4.19 Impact Factor
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    ABSTRACT: Cathepsin B (EC 3.4.22.1) is a lysosomal cysteine protease with both endopeptidase and exopeptidase activity. The former is associated with the degradation of the extracellular matrix proteins, which is a process required for tumour cell invasion and metastasis. In the present study, we show that 2A2 monoclonal antibody, raised by our group, is able to regulate cathepsin B activity. The EPGYSP sequence, located between amino acid residues 133-138 of cathepsin B in the proximity of the occluding loop, was determined to be the epitope for 2A2 monoclonal antibody using SPOT analysis. By surface plasmon resonance, an equilibrium dissociation constant (Kd) of 4.7 nM was determined for the interaction between the nonapeptide CIAEPGYSP, containing the epitope sequence, and 2A2 monoclonal antibody. 2A2 monoclonal antibody potentiated cathepsin B exopeptidase activity with a activation constant (Ka) of 22.3 nM, although simultaneously inhibiting its endopeptidase activity. The median inhibitory concentration values for the inhibition of hydrolysis of protein substrates, BODIPY FL casein and DQ-collagen IV were 761 and 702 nM, respectively. As observed by native gel electrophoresis and gel filtration, the binding of 2A2 monoclonal antibody to the cathepsin B/cystatin C complex caused the dissociation of cystatin C from the complex. The results obtained in the present study suggest that, upon binding, the 2A2 monoclonal antibody induces a conformational change in cathepsin B, stabilizing its exopeptidase conformation and thus disabling its harmful action associated with its endopeptidase activity.
    FEBS Journal 08/2009; 276(17):4739-51. · 4.25 Impact Factor
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    Janko Kos, Zala Jevnikar, Natasa Obermajer
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    ABSTRACT: Cathepsin X is a lysosomal cysteine protease, found predominantly in cells of monocyte/macrophage lineage. It acts as a monocarboxypepidase and has a strict positional and narrower substrate specificity relative to the other human cathepsins. In our recent studies we identified-beta(2) subunit of integrin receptors and alpha and gamma enolase as possible substrates for cathepsin X carboxypeptidase activity. In both cases cathepsin X is capable to cleave regulatory motifs at C-terminus affecting the function of targeted molecules. We demonstrated that via activation of beta(2) integrin receptor Mac-1 (CD11b/CD18) active cathepsin X enhances adhesion of monocytes/macrophages to fibrinogen and regulates the phagocytosis. By activation of Mac-1 receptor cathepsin X may regulate also the maturation of dendritic cells, a process, which is crucial in the initiation of adaptive immunity. Cathepsin X activates also the other beta(2) integrin receptor, LFA-1 (CD11a/CD18) which is involved in the proliferation of T lymphocytes. By modulating the activity of LFA-1 cathepsin X causes cytoskeletal rearrangements and morphological changes of T lymphocytes enhancing ameboid-like migration in 2-D and 3-D barriers and increasing homotypic aggregation. The cleavage of C-terminal amino acids of alpha and gamma enolase by cathepsin X abolishes their neurotrophic activity affecting neuronal cell survival and neuritogenesis.
    Cell adhesion & migration 05/2009; 3(2):164-6. · 2.34 Impact Factor
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    ABSTRACT: Membrane nanotubes were recently described as a new principle of cell-cell communication enabling complex and specific messaging to distant cells. Calcium fluxes, vesicles, and cell-surface components can all traffic between cells connected by nanotubes. Here we report for the first time the mechanism of membrane nanotube formation in T cells through LFA-1 (CD11a/CD18; alpha(L)beta(2)) integrin activation by the cysteine protease cathepsin X. Cathepsin X is shown to induce persistent LFA-1 activation. Cathepsin X-upregulated T cells exhibit increased homotypic aggregation and polarized, migration-associated morphology in 2D and 3D models, respectively. In these cells, extended uropods are frequently formed, which subsequently elongate to nanotubes connecting T lymphocytes. Our results demonstrate that LFA-1 activation with subsequent cytoskeletal reorganization induces signal transmission through a physically connected network of T lymphocytes for better coordination of their action at various stages of the immune response.
    Cellular and Molecular Life Sciences CMLS 03/2009; 66(6):1126-34. · 5.62 Impact Factor
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    ABSTRACT: Cathepsin X is a lysosomal cysteine protease exhibiting carboxypeptidase activity. Its expression is high in the cells of immune system and its function has been related to the processes of inflammatory and immune responses. It regulates processes such as adhesion, T lymphocyte activation and phagocytosis through its interaction with beta2 integrins. To investigate the role of cathepsin X in the migration of T lymphocytes, Jurkat T lymphocytes were stably transfected with a pcDNA3 expression vector containing cathepsin X cDNA. The cathepsin-X-overexpressing T lymphocytes exhibited polarised migration-associated morphology, enhanced migration on 2D and 3D models using intercellular adhesion molecule 1 (ICAM1)- and Matrigel-coated surfaces, and increased homotypic aggregation. The increased invasiveness of cathepsin-X-overexpressing cells does not involve proteolytic degradation of extracellular matrix. Confocal microscopy showed that the active mature form of cathepsin X was colocalised in migrating cells together with lymphocyte-function-associated antigen 1 (LFA-1). The colocalisation was particularly evident at the trailing edge protrusion, the uropod, that has an important role in T lymphocyte migration and cell-cell interactions. We propose that cathepsin X causes cytoskeletal rearrangements and stimulates migration of T lymphocytes by modulating the activity of the beta2 integrin receptor LFA-1.
    Journal of Cell Science 09/2008; 121(Pt 16):2652-61. · 5.88 Impact Factor