Summary
γ‐Enolase is a neurotrophic‐like factor promoting growth, differentiation, survival and regeneration of neurons. Its neurotrophic activity is regulated by cysteine protease cathepsin X which ...cleaves the C‐terminal end of the molecule. We have investigated the expression and colocalization of γ‐enolase and cathepsin X in brains of Tg2576 mice overexpressing amyloid precursor protein. In situ hybridization of γ‐enolase and cathepsin X revealed that mRNAs for both enzymes were expressed abundantly around amyloid plaques. Immunostaining demonstrated that the C‐terminally cleaved form of γ‐enolase was present in the immediate plaque vicinity, whereas the intact form, exhibiting neurotrophic activity, was observed in microglia cells in close proximity to senile plaque. The upregulation of γ‐enolase in microglial cells in response to amyloid‐β peptide (Aβ) was confirmed in mouse microglial cell line EOC 13.31 and primary microglia and medium enriched with γ‐enolase proved to be neuroprotective against Aβ toxicity; however, the effect was reversed by cathepsin X proteolytic activity. These results demonstrate an upregulation of γ‐enolase in microglia cells surrounding amyloid plaques in Tg2576 transgenic mice and demonstrate its neuroprotective role in amyloid‐β‐related neurodegeneration.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Cathepsin X (CTSX, also called cathepsin Z/P) is a cysteine protease that still plays an unknown role in human cancer. It has been shown to bind cell surface heparin sulphate proteoglycans and ...integrins, indicating possible functions of CTSX in cellular adhesion, phagocytosis, and immune response. Our previous studies have shown an association between Helicobacter pylori (H. pylori) infection, a strong up-regulation of CTSX, and development of gastric cancer. In this study, yeast two-hybrid analysis revealed that RPLP0, a ribosomal protein P0, interacts with the human CTSX protein in gastric cancer. The CTSX/RPLP0 interaction was confirmed by co-immunoprecipitation assays. In addition, co-localization studies in cancer cell line N87 and gastric cancer tissue samples were performed. Laserscan microscopy revealed a shuttling of RPLP0 (and CTSX) from cytoplasm to the nucleus after CTSX knockdown. Down-regulation of RPLP0 resulted in G1 arrest of gastric cancer cells, whereas knockdown of CTSX led to G1 arrest and apoptosis after 48h. Knockdown of both proteins caused increased apoptosis. RPLP0 deficiency could suppress cell growth and cell cycle progression by down-regulating CDK2. It was further demonstrated that RPLP0 affected p21 expression, but did not change the expression of Cyclin E. Down-regulation of both proteins at least through CDK2 suggests an anti-apoptotic effect on gastric cancer cells and opens up new possibilities for apoptotic immune modulation and gastric cancer therapy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
•We synthesized O,N-carboxymethyl chitosan–peptide–melphalan conjugates.•We studied in vitro drug release of O,N-carboxymethyl chitosan–peptide–melphalan conjugates.•These O,N-carboxymethyl ...chitosan–peptide–melphalan conjugates have enzyme-responsive characteristic.•The novel O,N-carboxymethyl chitosan–peptide–melphalan conjugates may be used as a new polymeric prodrug.
The clinical application of melphalan (Me), an anticancer drug for the treatment of hematologic malignancies, has been limited due to its poor water solubility, rapid elimination and lack of target specificity. To solve these problems, O,N-carboxymethyl chitosan–peptide–melphalan conjugates were synthesized and characterized. All polymeric prodrugs showed satisfactory water solubility. It was found that the molecular weight of O,N-carboxymethyl chitosan (O,N-CMCS) and the peptide spacer played a crucial role in controlling the drug content, diameter and drug release properties of O,N-carboxymethyl chitosan–peptide–melphalan conjugates. The studies of in vitro drug release and cell cytotoxicity by MTT assay revealed that, employing the polymeric conjugation strategy and using the peptides glycylglycine (Gly-Gly) as a spacer, the conjugates have good cathepsin X-sensitivity and lower toxicity and the drug release behavior improved remarkably. In conclusion, O,N-carboxymethyl chitosan–peptide–melphalan conjugates could be promising prodrugs for anticancer application.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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.
Understanding the molecular events involved in the acquisition of competence during oogenesis is a key step to determine the secret of ‘high quality’ eggs for aquaculture. Quantitative real time ...polymerase chain reaction (qPCR) is the technique of election to determine changes in transcript abundance in such studies, but choosing reference genes for normalization, in particular during oogenesis, remains a challenge.
In the present study, transcription of 6 functionally distinct genes, β actin (ACTB), cathepsin D (CTSD), cathepsin Z (CTSZ), elongation factor 1 α (EEF1A), TATA binding protein (TBP) and tubulin A (TUBA1A) was assessed as normalizers of bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI) gene expression in mRNA from Mozambique tilapia oocytes during oogenesis. Reverse transcription was equally efficient and varies little in all samples. Most of the genes considered for reference were stable during early stages of oogenesis but variations were observed during vitellogenesis. A single gene and up to 3 genes were shown to be insufficient for reliable normalization throughout the whole oogenesis. The combination of the genes ACTB, CTSD, EEF1A and CTSZ as reference was found to minimize variation and has the most stable expression pattern between maturation stages.
► Candidate reference genes were evaluated for expression stability during oogenesis. ► A 4-gene combination, ACTB, CTSD, EEF1A, and CTSZ, was assessed as normalizer. ► BAMBI expression in ovarian follicles decreased during vitellogenesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Cathepsin X is a cysteine protease involved in mechanisms of malignant progression. It is secreted from tumour cells as a proenzyme and may serve to predict the disease status and risk of death for ...cancer patients. In a previous, pilot, study on 77 colorectal patients we demonstrated the correlation of higher serum levels with shorter overall survival.
264 patients with colorectal cancer were included in a prospectively accrued multi-centre observational cohort study with the aim of testing novel biomarkers. Blood samples were collected before preoperative large bowel endoscopy and total cathepsin X was measured in sera by ELISA. As a control group we selected at random 77 subjects who had no findings at endoscopy and reported no co-morbidity.
The mean level of cathepsin X in cancer patients did not differ from the control levels (23.4 ng/ml ± 6.4 SD vs. 18.8 ng/ml ± 11.4 SD, p > 0.05) and there was no association with age, gender, disease stage, tumour location or CEA. In univariate analysis no association between cathepsin X levels and overall survival was demonstrated for the entire set of patients, however, cathepsin X was associated with survival in a group of patients with local resectable disease (stages I-III) (HR = 1.69, 95% CI: 1.03-2.75, p = 0.03). For this group, multivariate Cox regression analysis showed an association (HR = 3.13, 95% CI: 1.37-7.18, p = 0.003) between high cathepsin X levels and shorter overall survival for patients who did not receive chemotherapy, whereas, for patients who received chemotherapy, there was no association between cathepsin X and survival (HR = 0.51, 95% CI: 0.20-1.33, p = 0.88).
Association of cathepsin X levels with overall survival was not confirmed for an entire set of 264 colorectal patients, but for patients in stages I-III with local resectable disease. The significant association of cathepsin X with survival in a group of patients who received no chemotherapy and the absence of this association in the group who received chemotherapy, suggest the possible predictive value for response to chemotherapy. The results have to be confirmed in a further prospective study.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic neurons in the substantia nigra pars compacta (SNc).
, a contribution to neuroinflammation and ...neurotoxicity has been shown for the lysosomal protease cathepsin X; however, its expression and its role in PD remain unknown. Therefore, the current study was designed to address the regional, cellular, and subcellular localization and activity of cathepsin X in hemi-parkinsonian rats with 6-hydroxydopamine (6-OHDA)-induced excitotoxicity in the unilateral medial forebrain bundle (MFB) lesion. We report for the first time that cathepsin X expression and activity are rapidly increased in the ipsilateral SNc after injection of 6-OHDA into the MFB reaching a maximum after 12 h but seem to stay strongly upregulated after 4 weeks after injection. At early time points of 6-OHDA injection into the MFB, the increased cathepsin X is localized in the lysosomes in the neuronal, predominantly tyrosine hydroxylase-positive dopaminergic cells. After 12 h of 6-OHDA induced lesion, only a few activated microglial cells are positive for cathepsin X whereas, in 4 weeks post-lesion accompanied with complete loss of dopaminergic neurons, there is persistent cathepsin X upregulation restricted to activated glia cells. Taken together, our results demonstrate that cathepsin X upregulation in the lesioned dopaminergic system may play a role as a pathogenic factor in PD. Moreover, inhibition of cathepsin X expression or activity may be useful in protecting the nigrostriatal dopaminergic projection in the PD.
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 β₂ chain of LFA-1. Cleavage by cathepsin X of the amino acid residues S⁷⁶⁹, E⁷⁶⁸ and A⁷⁶⁷ from the C-terminal of the β₂ cytoplasmic tail of LFA-1 is shown to promote binding of the actin-binding protein α-actinin-1. Furthermore, cathepsin X overexpression reduced LFA-1 clustering and induced an intermediate affinity LFA-1 conformation that is known to associate with α-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.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Cathepsin X, a recently discovered lysosomal cysteine protease, shares common structural features and activity properties with cysteine protease cathepsin B. Based on its widespread mRNA distribution ...in primary tumors and tumor cell lines, a redundant function in tumor progression has been proposed. In this study, we have shown that these two related proteases exhibit different profiles with respect to their protein distribution in cells and tissues and to their possible roles in malignancy. Protein level of cathepsin X did not differ significantly between matched pairs of lung tumor and adjacent lung tissue obtained from patients with lung cancer whereas that of cathepsin B was 9.6-fold higher in tumor compared to adjacent lung tissue. Immunohistochemical analysis of lung tumor cathepsin X revealed very faint staining in tumor cells but positive staining in infiltrated histiocytes, alveolar macrophages, bronchial epithelial cells, and alveolar type II cells. Cathepsin X stained positive also in CD68
+ cells in germinal centers of secondary follicles in lymph nodes, corresponding to tingible body macrophages. Two cell lines with proven invasive behavior, MCF-10A neoT and MDA-MB 231, showed positive staining for cathepsin B, but negative for cathepsin X. We showed that the invasive potential of MCF-10A neoT cells can be impaired by specific inhibitor of cathepsin B but not by that of cathepsin X. Cathepsin X was found in large amounts in the pro-monocytic U-937 cell line, in monocytes and in dendritic cells, generated from monocytes in vitro. Our results show that cathepsin X is not involved in degradation of extracellular matrix, a proteolytic event leading to tumor cell invasion and metastasis. Its expression, restricted to immune cells suggests a role in phagocytosis and the regulation of immune response.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Up-regulation of lysosomal cysteine protease cathepsin X (Cat X) is associated with disorders of the immune system and neurodegenerative diseases, while its role in the development and progression of ...cancer is less understood. Enhanced secretion of pro-Cat X was observed in malignant processes, and therefore, the level of total serum Cat X rather than the active enzyme may better reflect the tumour status.
Seventy-seven patients with colorectal cancer (CRC) were included in a retrospective study. Blood samples were collected prior to therapy. Using ELISA, the values of total Cat X were measured in serum. Groups of healthy persons (n=77), patients with adenomas (n=77) and patients with non-neoplastic findings (n=77) were included.
Significant differences between the group of colorectal patients and the groups of healthy persons, adenoma patients and patients with non-malignant findings could not be shown (p=0.89). Within the group of CRC, higher levels of total Cat X significantly correlated to shorter overall survival (HR=2.08, 95% CI:1.07-4.05, p=0.028).
Total serum Cat X could be a useful prognostic indicator for determining survival of patients with CRC. Increased serum levels of total Cat X may reflect more aggressive tumour cell phenotypes and suggest the involvement of Cat X in processes involved in later stages of tumour progression.
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