New multienzymatic complex formed between human cathepsin D and snake venom phospholipase A2

Background Cathepsin D (CatD) is a lysosomal proteolytic enzyme expressed in almost all tissues and organs. This protease is a multifunctional enzyme responsible for essential biological processes such as cell cycle regulation, differentiation, migration, tissue remodeling, neuronal growth, ovulation, and apoptosis. The overexpression and hypersecretion of CatD have been correlated with cancer aggressiveness and tumor progression, stimulating cancer cell proliferation, fibroblast growth, and angiogenesis. In addition, some studies report its participation in neurodegenerative diseases and inflammatory processes. In this regard, the search for new inhibitors from natural products could be an alternative against the harmful effects of this enzyme. Methods An investigation was carried out to analyze CatD interaction with snake venom toxins in an attempt to find inhibitory molecules. Interestingly, human CatD shows the ability to bind strongly to snake venom phospholipases A2 (svPLA2), forming a stable muti-enzymatic complex that maintains the catalytic activity of both CatD and PLA2. In addition, this complex remains active even under exposure to the specific inhibitor pepstatin A. Furthermore, the complex formation between CatD and svPLA2 was evidenced by surface plasmon resonance (SPR), two-dimensional electrophoresis, enzymatic assays, and extensive molecular docking and dynamics techniques. Conclusion The present study suggests the versatility of human CatD and svPLA2, showing that these enzymes can form a fully functional new enzymatic complex.

Immunohistochemical Analysis of P-gp, LC3-II, and Cathepsin-D Associated with Histological Changes in Fish Liver: from the Impacted Environment to Clean Water

Abstract Herein we evaluated the histopathological alterations and expression patterns of multixenobiotic resistence (MXR) and autophagic proteins in liver samples of fish chronically exposed to anthropogenic contaminants in a highly polluted river, and then again after they had been transferred to good quality water. Two groups were established: euthanized on the day of capture (0 h), and maintained for 30 days in a tank (30 d). The fish of 0 h presented liver with vacuolated and hypertrophic hepatocytes. Also, it was observed strong immunostaining of cathepsin-D, LC3-II and P-gp. Necrosis and apoptosis were also observed throughout the liver. Conversely, the second group (30 d) showed recovery of the liver normal histology and weak immunoreaction of the studied proteins. So, our results indicated that there was a hepatic recovery in the fish kept in good quality water, as showed by the decreased expression of cathepsin-D, LC3-II, and the MXR (P-gp). Therefore, the alterations here observed could be proposed as potential biomarkers to be tested for following the impacts of remediation or mitigation measures to environmental impacts.

The Expression of Multiple Proteins as Prognostic Factors in Colorectal Cancer: Cathepsin D, p53, COX-2, Epidermal Growth Factor Receptor, C-erbB-2, and Ki-67

BACKGROUND/AIMS: A single gene mutation alone cannot explain the poor prognosis of colorectal cancer. This study aimed to establish a correlation between the expression of six proteins and the prognosis of colorectal cancer patients. METHODS: Tissue samples were collected from 266 patients who underwent surgery for colorectal cancer at our institution from January 2006 to December 2007. The expression of six proteins were determined using immunohistochemical staining of specimens. RESULTS: Cathepsin D, p53, COX-2, epidermal growth factor receptor, c-erbB-2, and Ki-67 expression were detected in 38.7%, 60.9%, 37.6%, 35.7%, 30.1%, and 74.4% of the samples, respectively. The expression of cathepsin D was significantly correlated with reduced cancer-free survival (p=0.036) and colorectal cancer-specific survival (p=0.003), but the other expression levels were not. In a multivariate analysis, cathepsin D expression was found to be an independent prognostic factor for poorer colorectal cancer-specific survival (hazard ratio, 8.55; 95% confidence interval, 1.07 to 68.49). Furthermore, patients with tumors expressing four or more of the proteins had a significantly decreased cancer-free survival rate (p=0.006) and colorectal cancer-specific survival rate (p=0.002). CONCLUSIONS: Patients with cathepsin D positivity had a poorer outcome than patients who were cathepsin D-negative. Thus, cathepsin D may provide an indicator for appropriate intensive follow-up and adjuvant chemotherapy.

Expresión de catepsina D en carcinoma mamario y sus correlaciones clínicas e histopatológicas / Cathepsin D expression in breast cancer and its correlations with clinical and histopathological parameters

Antecedentes. La catepsina D es una proteasa lisosomal que se puede sobrexpresar en cáncer mamario. Varios estudios hechos en citosol de tejido tumoral demuestran que los niveles elevados de catepsina D se asocian con pronóstico desfavorable en pacientes con cáncer mamario pero en estudios realizados por inmunohistoquímica, los resultados no son concluyentes. Objetivo. Evaluar si catepsina D, medida por técnica inmunohistoquímica con un anticuerpo policlonal, puede ser considerada como factor pronóstico en el cáncer de mama. Pacientes y métodos. Se realizó en 68 muestras tumorales que correspondían a pacientes con cáncer mamario en etapas clínicas I-IV, tratadas en el Instituto Nacional de Cancerología durante los años 1985 y 1986. Resultados. Treinta y cinco de las 68 pacientes (51 por ciento) presentaron tinción positiva intensa para catepsina D, 19 (28 por ciento) tuvieron tinción leve y 14 (21 por ciento) fueron negativas. En 10 de las pacientes con tinción leve existieron artificios por defectos en la fijación del tejido. La expresión de catepsina D no tuvo valor pronóstico ni se encontró asociación de la catepsiana D con otros factores pronósticos clínicos e histopatológicos conocidos. Conclusión. La catepsina D determinada por inmunohistoquímica no tuvo valor pronóstico en el cáncer de mama

Cytosol cathepsin D in Thai women with breast cancer.

The necessity of screening high-risk patients with breast cancer who may require more intensive systemic therapy especially in the node negative subgroup was generally accepted. Cathepsin D, an estrogen induced protease, has been shown to be implicated in the proliferation and invasion of breast cancers. Retrospective assessment of cytosol cathepsin D in 151 primary breast cancers was done together with ER, PR and other clinico-pathological parameters. No significant relationship was shown between cathepsin D concentrations or cathepsin D status using median value of 56 pmol/mg protein as cutoff level with most studied parameters. High cathepsin D status was found in 47 per cent of patients with fibrocystic disease of breast and 30 per cent in node-negative, ER-PR negative tumors. Survival analysis after 5 or 10 year follow-up and evaluation in a larger scale are necessary before including cytosol cathepsin D measurement as a routine clinical investigation for breast cancers.

Activation of protease activity in rat peritoneal macrophages in protein deficiency: characterization of cathepsin D.

Rat peritoneal macrophages contained high proteolytic activity that was significantly enhanced under the stress induced by protein deficiency. The aspartyl protease cathepsin D which has been known to be the most active protease in endocytic processes was extracted from the macrophages recovered from control (20% protein fed) and protein deficient (4% protein fed) rats and was affinity purified and characterized further. The cathepsin D from the control sample exhibited better recovery, purification and higher specific activity compared to that from the deficient groups. Apparently the pH optima and heat stability of the enzyme from both the groups were similar. The SDS PAGE profile clearly indicated the presence of greater amounts of active forms of cathepsin D in the deficient samples in vivo itself which reflected in a reduction in Km value of the enzyme. Subtle differences observed in the activity of these macrophage proteases in the protein deficient rats may be partly responsible for the enhanced degradation of macrophage membrane proteins reported earlier.