Increased cell surface protease activity in UV-irradiated cells undergoing apoptosis

Abstract

UVB-irradiated HeLa cells undergoing apoptosis have increased cell surface protease (CSP) activity compared to viable or necrotic cells. In order to elucidate whether caspase 3 plays a role in the activation of CSP in cells undergoing apoptosis, HeLa cell cultures were pre-treated with the caspase inhibitor, DEVD, prior to being exposed to 500 Jm^-2 UVB. DEVD significantly inhibited caspase 3 activity in cells undergoing apoptosis, but did not affect the activation of CSP in these cells. The findings suggest that the activation of CSP in apoptotic cells is unrelated to caspase 3 activity.     

Increased cell surface protease activity in UV-irradiated cells undergoing apoptosis

UVB-irradiated HeLa cells undergoing apoptosis have increased cell surface protease (CSP) activity compared to viable or necrotic cells. In order to elucidate whether caspase 3 plays a role in the activation of CSP in cells undergoing apoptosis, HeLa cell cultures were pre-treated with the caspase inhibitor, DEVD, prior to being exposed to 500 Jm(-2) UVB. DEVD significantly inhibited caspase 3 activity in cells undergoing apoptosis, but did not affect the activation of CSP in these cells. The findings suggest that the activation of CSP in apoptotic cells is unrelated to caspase 3 activity.     

HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins

Inhibitor of apoptosis (IAP) proteins inhibit caspases, a function counteracted by IAP antagonists, insect Grim, HID, and Reaper and mammalian DIABLO/Smac. We now demonstrate that HtrA2, a mammalian homologue of the Escherichia coli heat shock-inducible protein HtrA, can bind to MIHA/XIAP, MIHB, and baculoviral OpIAP but not survivin. Although produced as a 50-kDa protein, HtrA2 is processed to yield an active serine protease with an N terminus similar to that of Grim, Reaper, HID, and DIABLO/Smac that mediates its interaction with XIAP. HtrA2 is largely membrane-associated in healthy cells, with a significant proportion observed within the mitochondria, but in response to UV irradiation, HtrA2 shifts into the cytosol, where it can interact with IAPs. HtrA2 can, like DIABLO/Smac, prevent XIAP inhibition of active caspase 3 in vitro and is able to counteract XIAP protection of mammalian NT2 cells against UV-induced cell death. The proapoptotic activity of HtrA2 in vivo involves both IAP binding and serine protease activity. Mutations of either the N-terminal alanine of mature HtrA2 essential for IAP interaction or the catalytic serine residue reduces the ability of HtrA2 to promote cell death, whereas a complete loss in proapoptotic activity is observed when both sites are mutated.     

Relationship between cell surface protease activity and doubling time in various normal and transformed cells

A sensitive method for measuring cell surface and secreted protease activity utilizing ³H-labelled casein is described. The method is based upon proteolytic degradation of the casein substrate into trichloroacetic acid soluble ³H-labelled peptides. Utilizing the radioassay we found that all cultured cell lines examined contain cell surface proteolytic activity which is not secreted into the media. The protease activity was found to be due to protease(s) other than plasminogen activator or plasmin. A comparison of surface protease activity of normal and transformed mouse epidermal cells indicated that the transformed cells contained approximately 3–4 times more proteolytic activity than the normal cells.Surface protease activity was also correlated with the doubling times of various cultured cells. The results indicated that cultured cells with doubling times of greater than three days possess less surface protease activity than cells with shorter doubling times. In order to determine changes in the levels of surface protease activity during the cell cycle several cell lines were synchronized. In synchronized rabbit aortic fibroblasts, mouse transformed epidermal cells and human melanoma cells, a marked increase in surface protease activity was observed during or before mitosis. The protease levels decreased following mitosis. The results suggest that in culture, cell surface protease(s) may be important factor in regulating the rate of cell growth.     

Estimation of amoeba cell volume from nuclear diameter and its application to studies in protozoan ecology

To facilitate the estimation of cell volume in uninucleate, naked amoebae (gymnamoebae) the relationship, log cell volume (µm³) = 0.882 + 3.117log nuclear diameter (µm³), is presented. This links mean cell volume to mean nuclear diameter and provides a useful tool for protozoan ecologists interested in estimating the biovolume of amoebae in laboratory or field samples. While it is virtually impossible to measure rigid axes from which volume can be calculated in these amorphous cells, it is relatively easy to measure the diameter of the nucleus in living or fixed material. This relationship has shown that most uninucleate amoebae surveyed have volumes ranging between only 188 µm³ and 2860 µm³; this range reflects the volumes of the majority of amoebae in the field. These small volumes are unexpected since many amoebae have locomotive forms greater than 20 µm in length giving the impression that their cell volumes should be correspondingly large. This is not the case, however, because most amoebae are extremely flat when viewed in profile. The small cell volume of most amoeba species has ecological implications when numerical data is transformed to biovolume and biomass units.     

Relationship between cell surface protease activity and doubling time in various normal and transformed cells.

A sensitive method for measuring cell surface and secreted protease activity utilizing 3H-labelled casein is described. The method is based upon proteolytic degradation of the casein substrate into trichloracetic acid soluble 3H-labelled peptides. Utilizing the radioassay we found that all cultured cell lines examined contain cell surface proteolytic activity which is not secreted into the media. The protease activity was found to be due to protease(s) other than plasminogen activator or plasmin. A comparison of surface protease activity of normal and transformed mouse epidermal cells indicated that the transformed cells contained approximately 3--1 times more proteolytic activity than the normal cells. Surface protease activity was also correlated with the doubling times of various cultured cells. The results indicated that cultured cells with doubling times of greater than three days possess less surface protease activity than cells with shorter doubling times. In order to determine changes in the levels of surface protease activity during the cell cycle several cell lines were synchronized. In synchronized rabbit aortic fibroblasts, mouse transformed epidermal cells and human melanoma cells, a marked increase in surface protease activity was observed during or before mitosis. The protease levels decreased following mitosis. The results suggest that in culture, cell surface protease(s) may be important factor in regulating the rate of cell growth.     

The relationship between surface protease activity and the rate of cell proliferation in normal and transformed cells

Surface protease activity and secreted protease activity has been determined on several cell lines utilizing ³H-acetyl casein as substrate. When normal rabbit aortic smooth muscle cells and fibroblasts stopped proliferating at high cell density a decrease in surface protease activity was observed. No decrease in surface protease activity or in the rate of cell proliferation was observed in either transformed melanoma or epidermal cells. The decrease of surface protease activity was related to a decrease in cell proliferation.     

Cell surface energy and membrane associated actin in lymphocytes

We have shown previously that membrane associated actin correlates with the migratory abilities of lymphocytes during recirculation, and that cell surface energy correlates with the adhesiveness of lymphocytes to other cells. In this study, measurements of actin content and cell surface energy have been made for various lymphocyte subpopulations to examine the possibility that recirculation ability may be related to nonspecific adhesiveness. We have found that: both cell surface energy and actin content combine to provide a consistent explanation for the relative rates of recirculation of various lymphocyte subpopulations, and cell surface energies and actin contents vary independently in these lymphocyte subpopulations. Comparison of the actin contents and cell surface energies of metastatic and nonmetastatic lymphoma cell lines indicated that the differences in metastatic potential were more likely attributable to specific receptor-ligand interactions than to nonspecific adhesiveness. Cell surface energy and actin content are consistent with the greater adhesiveness of B cells than T cells to nylon wool, providing a physical basis for this common cell separation technique.     

Cell surface energy and membrane associated actin in lymphocytes

We have shown previously that membrane associated actin correlates with the migratory abilities of lymphocytes during recirculation, and that cell surface energy correlates with the adhesiveness of lymphocytes to other cells. In this study, measurements of actin content and cell surface energy have been made for various lymphocyte subpopulations to examine the possibility that recirculation ability may be related to nonspecific adhesiveness. We have found that: both cell surface energy and actin content combine to provide a consistent explanation for the relative rates of recirculation of various lymphocyte subpopulations, and cell surface energies and actin contents vary independently in these lymphocyte subpopulations. Comparison of the actin contents and cell surface energies of metastatic and nonmetastatic lymphoma cell lines indicated that the differences in metastatic potential were more likely attributable to specific receptor-ligand interactions than to nonspecific adhesiveness. Cell surface energy and actin content are consistent with the greater adhesiveness of B cells than T cells to nylon wool, providing a physical basis for this common cell separation technique.     

Evidence for inhibitors of the cell surface protease guanidinobenzoatase

Guanidinobenzoatase is a protease present on the surface of tumour cells. The present study describes the isolation of a protein inhibitor of guanidinobenzoatase obtained from extracts of liver and pancreas and purified by affinity techniques. Pancreatic acinar cells have been shown to possess a latent form of guanidinobenzoatase and this latency is due to complex formation with the inhibitor. A fluorescent marker has been employed to demonstrate the presence or absence of the inhibitor on sections of pancreatic tissue. The inhibitor has been shown to be exchangeable with liver and pancreatic inhibitors obtained from different species. It is postulated that these inhibitors may play a role in enzyme control.     

Heparin inhibits enhancing factor/phospholipase A2 activity and its binding to the cell surface

Enhancing factor (EF), a mouse intestinal phospholipase A₂ (PLA₂), has been isolated and characterized. EF increases the binding of epidermal growth factor (EGF) to A431 cells almost two-fold by interacting with EGF. EF binds to a 100 kDa cell surface receptor and brings about an increase in the binding of EGF. In the present study we demonstrate that EF is a heparin binding protein and at the time of iodination of EF, the heparin binding site of EF has to be protected. Heparin inhibits the enhancing activity of EF as well as the binding of labelled EF to A431 cells. Inhibition of binding of EF to cells by heparin indicates that heparin binding region forms at least part of the receptor binding domain. These data suggest that the receptor for EF on the cell surface could be a heparin sulphate proteoglycan.     

Posttranslational modifications of serine protease TMPRSS13 regulate zymogen activation,proteolytic activity,and cell surface localization

TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, harbors four N-linked glycosylation sites in its extracellular domain. Two of the glycosylated residues are located in the scavenger receptor cysteine-rich (SRCR) protein domain, while the remaining two sites are in the catalytic serine protease (SP) domain. In this study, we examined the role of N-linked glycosylation in the proteolytic activity, autoactivation, and cellular localization of TMPRSS13. Individual and combinatory site-directed mutagenesis of the glycosylated asparagine residues indicated that glycosylation of the SP domain is critical for TMPRSS13 autoactivation and catalytic activity toward one of its protein substrates, the prostasin zymogen. Additionally, SP domain glycosylation-deficient TMPRSS13 displayed impaired trafficking of TMPRSS13 to the cell surface, which correlated with increased retention in the endoplasmic reticulum. Importantly, we showed that N-linked glycosylation was a critical determinant for subsequent phosphorylation of endogenous TMPRSS13. Taken together, we conclude that glycosylation plays an important role in regulating TMPRSS13 activation and activity, phosphorylation, and cell surface localization.     

酵母细胞表面展示技术及其在非水相酶催化合成中的应用

酵母展示技术是将外源蛋白与酵母细胞壁蛋白融合,并将外源蛋白表达在酵母细胞表面。酵母展示技术已广泛应用于各种功能蛋白的表达及筛选。以下重点介绍酵母展示技术在脂肪酶展示体系构建及其在脂肪酸甲酯、短链芳香酯及糖酯生物合成中的应用。     

Effect of sulfur exposure on protease activity in human peripheral blood lymphocytes

Sulfur mustard is a waemical warfare blistering agent for which neither the mechanism of action nor an antidote is known. Papirmeister et al. (1985) have postulated a biochemical hypothesis for mustard-induced cutaneous injury involving a sequelae of DNA alkylation, metabolic disruption and activation of protease. Human peripheral blood lymphocytes in cell cultures were employed as an in vitro model for alkylating agent toxicity. A chromogenic peptide substrate assay was used for detection of protease in lymphocytes treated with sulfur mustard or chloroethyl ethyl sulfide. Exposure of human peripheral blood lymphocytes from normal donors to these alkylating agents resulted in an increase in cell associated protease activity. This increase in protease activity may contribute to the pathology or act as an indicator to predict methods of therapeutic intervention for sulfur mustard toxicity.Abbreviations PBL peripheral blood lymphocytes - CEES chloroethyl ethyl sulfide - DFP diisopropyl fluoro-phosphate - pNA p-nitroaniline - CPSPA Chromogenic Peptide Substrate Protease Assay The opinions or assertions herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.     

Alkaline protease activity associated with iridescent virus type 6

Abstract An alkaline protease activity has been found to be associated with Iridovirus type 6. The enzyme showed a pH optimum of 10–10.5 with azocoll and hemoglobin but was essentially inactive on casein. From inhibitor studies the enzyme behaved like a serine protease. An M _r value of about 11500 was determined by SDS-PAGE.     

Histidine-rich glycoprotein binds heparanase and regulates its enzymatic activity and cell surface interactions

Heparanase, an endo-β-d-glucuronidase, is involved in numerous normal physiological and pathological processes, such as inflammation, wound healing and tumour metastasis/angiogenesis, through its ability to mediate the degradation of heparan sulfate, a key structural component of the extracellular matrix and on the surface of cells. Identifying endogenous molecules that can regulate heparanase activity will aid the understanding of its molecular function in health and disease and provide the potential for development of novel anti-cancer and anti-inflammatory therapeutics. The ability of the extracellular heparanase to tether onto cell surface heparan sulfate proteoglycans and other receptor(s), such as the cation-independent mannose-6-phosphate receptor, is key to its activation, function and uptake into intracellular compartments. Here we describe experiments demonstrating that a relatively abundant plasma glycoprotein, histidine-rich glycoprotein, directly interacts with platelet-derived heparanase and enhances its enzymatic activity. The findings in this study also show that histidine-rich glycoprotein interferes with heparanase binding to cell surface receptors, particularly heparan sulfate proteoglycans. Thus, the interaction between histidine-rich glycoprotein and heparanase can potentially regulate the role of heparanase in a variety of physiological and pathological conditions.     

Contribution of a metal-peroxide adduct to neurodegeneration is due to its oxidative protease activity

Many hypotheses have been developed to explain aging and age-related neurodegenerative disorders; one of the most compelling is the role of oxidative stress to induce changes in protease activity in brains of patients of Alzheimer's disease and prion disease. At the moment however, there is no clear answer how protein degradation may be achieved in the brain. We have observed that several metal compounds can degrade proteins in the presence of hydrogen peroxide, and elucidated the reaction scheme based on the new theoretical point for the reactivity of a metal-peroxide adduct with eta 1-coordination mode. In this article we would like to point out the importance of a copper(II)-peroxide adduct to promote neurodegenerative diseases such as prion disease and amyotrophic lateral sclerosis through its oxidative protease function.