Zala Jevnikar

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.

The role of cathepsin X in the migration and invasiveness of T lymphocytes

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 β2 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 β2 integrin receptor LFA-1.

Cysteine protease cathepsin X modulates immune response via activation of β2 integrins

Cathepsin X is a lysosomal, cysteine dependent carboxypeptidase. Its expression is restricted to cells of the immune system, suggesting a function related to the processes of inflammatory and immune responses. It has been shown to stimulate macrophage antigen‐1 (Mac‐1) receptor‐dependent adhesion and phagocytosis via interaction with integrin β2 subunit. Here its potential role in regulating lymphocyte proliferation via Mac‐1 and the other β2 integrin receptor, lymphocyte function‐associated antigen‐1 (LFA‐1) has been investigated. Cathepsin X has been shown to suppress proliferation of human peripheral blood mononuclear cells, by activation of Mac‐1, known as a suppressive factor for lymphocyte proliferation. On the other hand, co‐localization of cathepsin X and LFA‐1 supports the role of cathepsin X in regulating LFA‐1 activity, which enhances lymphocyte proliferation. As shown by fluorescence resonance energy transfer, using U‐937 and Jurkat cells transfected with αL‐mCFP and β2‐mYFP, recombinant cathepsin X directly activates LFA‐1. The activation was confirmed by increased binding of monoclonal antibody 24, recognizing active LFA‐1. We demonstrate that cathepsin X is involved in the regulation of two β2 integrin receptors, LFA‐1 and Mac‐1, which exhibit opposing roles in lymphocyte activation.

Novel mechanism of cathepsin B inhibition by antibiotic nitroxoline and related compounds

A new trick for an old dog! Aberrant cathepsin B activity is associated with tumor progression, however, despite extensive research, there are no cathepsinB inhibitors in clinical use. Here, nitroxoline, an established antimicrobial agent, is identified as a potent, reversible inhibitor of cathepsin B, and is thus a potential drug candidate for the treatment of cancer and other diseases in which cathepsin B activity plays a role.

The role of cathepsin X in cell signaling

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  β2 subunit of integrin receptors and α and γ 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 β2 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 β2 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 α and γ enolase by cathepsin X abolishes their neurotrophic activity affecting neuronal cell survival and neuritogenesis.

Identification of Candidate Carrier Proteins for Surface Display on Lactococcus lactis by Theoretical and Experimental Analyses of the Surface Proteome

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.

Expression of the sweet-tasting plant protein brazzein in Escherichia coli and Lactococcus lactis: a path toward sweet lactic acid bacteria

Brazzein is an intensely sweet-tasting plant protein with good stability, which makes it an attractive alternative to sucrose. A brazzein gene has been designed, synthesized, and expressed in Escherichia coli at 30 °C to yield brazzein in a soluble form and in considerable quantity. Antibodies have been produced using brazzein fused to His-tag. Brazzein without the tag was sweet and resembled closely the taste of its native counterpart. The brazzein gene was also expressed in Lactococcus lactis, using a nisin-controlled expression system, to produce sweet-tasting lactic acid bacteria. The low level of expression was detected with anti-brazzein antibodies. Secretion of brazzein into the medium has not led to significant yield increase. Surprisingly, optimizing the codon usage for Lactococcus lactis led to a decrease in the yield of brazzein.

Iron‐regulated surface determinant B (IsdB) promotes Staphylococcus aureus adherence to and internalization by non‐phagocytic human cells

Staphylococcus aureus is a human pathogen that causes invasive and recurring infections. The ability to internalize into and persist within host cells is thought to contribute to infection. Here we report a novel role for the well‐characterized iron‐regulated surface determinant B (IsdB) protein which we have shown can promote adhesion of 293T, HeLa cells and platelets to immobilized bacteria independently of its ability to bind haemoglobin. IsdB bound to the active form of the platelet integrin αIIbβ3, both on platelets and when the integrin was expressed ectopically in CHO cells. IsdB also promoted bacterial invasion into human cells. This was clearly demonstrated with bacteria lacking fibronectin‐binding proteins (FnBPs), which are known to promote invasion in the presence of fibronectin. However, IsdB also contributed significantly to invasion by cells expressing FnBPs in the presence of serum. Thus IsdB appears to be able to interact with the broader family of integrins that bind ligands with the RGD motif and to act as a back up mechanism to promote interactions with mammalian cells.

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.

Three-dimensional invasion of macrophages is mediated by cysteine cathepsins in protrusive podosomes

Podosomes, specialized actin‐rich structures in macrophages (Mfs), degrade the extra‐cellular matrix (ECM) and are involved in cell migration. On two‐dimensional (2D) surfaces Mfs 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 Mfs and in human monocytic cell line U‐937. Monocyte‐to‐Mf 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 Mf 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 Mf 3D invasion, could be an approach to the treatment of inflammatory and cancerous diseases.