Proteolytic activities during growth and aging in the fungus Podospora anserina: Effect of specific mutations

In Podospora anserina five proteolytic enzymes were characterized by chromatographic procedures. Three of these (proteases A, B and C) were found in the cell extracts of growing cultures and the other two (proteases III and IV) were revealed by studies on protoplasmic incompatibility. During growth, only protease C, an acidic enzyme, was active in crude extracts. From the stationary and the poststationary stages this activity decreased and finally disappeared, whereas a neutral serine protease (activity B) became active in crude extracts. A close relationship was observed between the proteolytic activity of the culture filtrates and the intracellular protease(s) concomitantly active in the crude extracts. None of the proteases associated with protoplasmic incompatibility was detected, both in the extra- and intracellular spaces. Qualitative variations in the proteolytic activities during stationary and post-stationary stages depended on the presence of specific genes and mutations: the mod C mutation suppressing protoplasmic incompatibility, inhibits the progressive decrease of protease C and, furthermore, the presence of non allelic incompatibility genes have for consequence the substitution of serine protease B by serine protease A during the poststationary stage.     

Degradation of DNA topoisomerase I by a novel trypsin-like serine protease in proliferating human T lymphocytes

DNA topoisomerase I (Topo I) contributes to various important biological functions, and its activity is therefore likely regulated in response to different physiological conditions. Increases in both the synthesis and degradation of Topo I were previously shown to accompany phytohemagglutinin stimulation of proliferation in human peripheral T lymphocytes. The mechanism of this degradation of Topo I has now been investigated with both in vivo and in vitro assays. The activity of a nuclear protease that specifically degrades Topo I was induced in proliferating T lymphocytes. The full-length Topo I protein (100 kDa) was sequentially degraded to 97- and 82-kDa fragments both in vivo and in vitro. The initial site of proteolytic cleavage was mapped to the NH(2)-terminal region of the enzyme. The degradation of Topo I in vitro was inhibited by aprotinin or soybean trypsin inhibitor, suggesting that the enzyme responsible is a trypsin-like serine protease. Furthermore, Topo I degradation by this protease was Mg(2+)-dependent. The Topo I-specific protease activity induced during T lymphocytes proliferation was not detected in Jurkat (human T cell leukemia) cells and various other tested human cancer cell lines, possibly explaining why the abundance of Topo I is increased in tumor cells.     

Human seminal vesicle-specific antigen is a substrate for prostate-specific antigen (or P-30)

Human seminal vesicle and prostatic fluids were obtained separately and reconstituted in vitro to test the hypothesis that proteolytic enzymes of prostatic origin would degrade seminal vesicle-specific antigen (SVSA). Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis to follow the fate of SVSA over time, we found that upon mixing the two secretions, SVSA was converted to forms of intermediate and low molecular weight identical to transformations seen in normal liquefied ejaculates. Diisoproprylfluorophophate, a serine protease inhibitor, prevented this degradation, indicating serine protease involvement in the proteolysis of SVSA. Prostate-specific antigen (PSA; also known as P-30), recently identified as a serine protease, was examined for its ability to mimic the effects of prostatic fluid on SVSA. Purified PSA catalyzed degradation of SVSA to produce proteolytic fragments that comigrated and were immunologically related to SVSA fragments produced by prostatic fluid. Purified PSA in the presence of serine protease inhibitors was unable to degrade SVSA. These results demonstrate that SVSA is a substrate for PSA during human semen liquefaction.     

Characterization and isolation of a trypsin-like serine protease from a long-term culture cytolytic T cell line and its expression by functionally distinct T cells

We describe the characterization and purification of a trypsin-like serine protease isolated from cloned long-term culture cytolytic T cell line (CTLL AK). High amounts of proteolytic activity were isolated from extracts of CTLL AK after either nitrogen cavitation or detergent lysis. Trypsin-like protease was detected by using either the ester compound N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester or a panel of low molecular amide substrates. The latter compounds were preferentially cleaved at the carboxyl termini of lysine and arginine residues. The enzyme activity was completely inhibited by two serine esterase inhibitors, diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and by aprotinin and meta-aminobenzamidine, which are known to block trypsin-like proteases. The pH optimum for CTLL AK-derived protease activity is 8 to 9. Analysis of the enzyme by gel filtration revealed that the cell-bound proteolytic activity was associated with a complex that could not be dissociated by treatment with Triton X-100. The CTLL AK-derived protease activity was found to reside in two proteins with relative molecular masses (Mr) of 32,000 and 40,000 daltons as determined by affinity labeling with [3H]diisopropylfluorophosphate and sodium dodecyl sulfate gel electrophoresis. High levels of enzyme activity were found in a panel of H-Y-specific cloned T cell lines with either cytolytic/suppressor (CTLL) or helper potential (THL), indicating a lack of correlation between trypsin-like protease activity and a particular T cell function. High enzyme activity was also detected in tumorigenic variants of CTLL. Furthermore, it was excluded that the trypsin-like activity detected was attributable to plasminogen activator activity. In contrast to cloned T effector cells and their in vitro or in vivo derived variants, considerably less activity was found in normal nonactivated or activated lymphocyte populations. The possible role of the trypsin-like serine protease in the function of T effector cells is discussed.     

A soluble ATP-dependent system for protein degradation from murine erythroleukemia cells. Evidence for a protease which requires ATP hydrolysis but not ubiquitin

A soluble ATP-dependent system for protein degradation has been demonstrated in reticulocyte lysates, but not in extracts of nucleated cells. We report that extracts of undifferentiated murine erythroleukemia (MEL) cells contain a labile ATP-stimulated proteolytic system. The addition of ATP to MEL cell extracts at alkaline pH enhances degradation of endogenous cell proteins and various radiolabeled exogenous polypeptides from 2-15-fold. Nonhydrolyzable ATP analogs had no effect. In reticulocytes, one role of ATP in proteolysis is for ubiquitin conjugation to protein substrates. MEL cells also contain ubiquitin and extracts can conjugate 125I-ubiquitin to cell proteins; however, this process in MEL cells seems unrelated to protein breakdown. After removal of ubiquitin from these extracts by DEAE- or gel chromatography, the stimulation of proteolysis by ATP was maintained and readdition of purified ubiquitin had no further effect. In addition, these extracts degraded in an ATP-dependent fashion casein whose amino groups were blocked and could not be conjugated to ubiquitin. After gel filtration or DEAE-chromatography of the MEL cell extracts (unlike those from reticulocytes), we isolated a high molecular weight (600,000) ATP-dependent proteolytic activity, which exhibits many of the properties of energy-dependent proteolysis seen in crude cell extracts. For example, both the protease and crude extracts are inhibited by hemin and N-ethylmaleimide and both hydrolyze casein, globin, and lysozyme rapidly and denatured albumin relatively slowly. The protease, like the crude extracts, is also stimulated by UTP, CTP, and GTP, although not as effectively as ATP. Also, nonhydrolyzable ATP analogs and pyrophosphate do not stimulate the protease. Thus, some mammalian cells contain a cytosolic proteolytic pathway that appears independent of ubiquitin and involves and ATP-dependent protease, probably similar to that found in Escherichia coli or mitochondria.     

The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain

Enzymes of the ATP-independent Deg serine endopeptidase family are very flexible with regard to their substrate specificity. Some family members cleave only one substrate, while others act as general proteases on unfolded substrates. The proteolytic activity of Deg proteases is regulated by PDZ protein interaction domains. Here we characterized the HhoA protease from Synechocystis sp. strain PCC 6803 in vitro using several recombinant protein constructs. The proteolytic activity of HhoA was found to increase with temperature and basic pH and was stimulated by the addition of Mg(2+) or Ca(2+). We found that the single PDZ domain of HhoA played a critical role in regulating protease activity and in the assembly of a hexameric complex. Deletion of the PDZ domain strongly reduced proteolysis of a sterically challenging resorufin-labeled casein substrate, but unlabeled beta-casein was still degraded. Reconstitution of the purified HhoA with total membrane proteins isolated from Synechocystis sp. wild-type strain PCC 6803 and a DeltahhoA mutant resulted in specific degradation of selected proteins at elevated temperatures. We concluded that a single PDZ domain of HhoA plays a critical role in defining the protease activity and oligomerization state, combining the functions that are attributed to two PDZ domains in the homologous DegP protease from Escherichia coli. Based on this first enzymatic study of a Deg protease from cyanobacteria, we propose a general role for HhoA in the quality control of extracytoplasmic proteins, including membrane proteins, in Synechocystis sp. strain PCC 6803.     

Intracellular proteolysis of pancreatic zymogens.

Activation of pancreatic digestive zymogens within the pancreatic acinar cell may be an early event in the development of pancreatitis. To detect such activation, an immunoblot assay has been developed that measures the relative amounts of inactive zymogens and their respective active enzyme forms. Using this assay, high doses of cholecystokinin or carbachol were found to stimulate the intracellular conversion of at least three zymogens (procarboxypeptidase A1, procarboxypeptidase B, and chymotrypsinogen 2) to their active forms. Thus, this conversion may be a generalized phenomenon of pancreatic zymogens. The conversion is detected within ten minutes of treatment and is not associated with changes in acinar cell morphology; it has been predicted that the lysosomal thiol protease, cathepsin B, may initiate this conversion. Small amounts of cathepsin B are found in the secretory pathway, and cathepsin B can activate trypsinogen in vitro; however, exposure of acini to a thiol protease inhibitor (E64) did not block this conversion. Conversion was inhibited by the serine protease inhibitor, benzamidine, and by raising the intracellular pH, using chloroquine or monensin. This limited proteolytic conversion appears to require a low pH compartment and a serine protease activity. After long periods of treatment (60 minutes), the amounts of the active enzyme forms began to decrease; this observation suggested that the active enzyme forms were being degraded. Treatment of acini with E64 reduced this late decrease in active enzyme forms, suggesting that thiol proteases, including lysosomal hydrolases, may be involved in the degradation of the active enzyme forms. These findings indicate that pathways for zymogen activation as well as degradation of active enzyme forms are present within the pancreatic acinar cell.     

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Specific Proteolysis in Barley Chloroplasts During Dark Induced Senescence

Intact chloroplasts were isolated from dark-senescing primary barley (Hordeum vulgare L.) leaves in order to study selective ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) degradation by the stromal and membrane fractions. RuBPCO specific degradation was estimated and characterised applying sensitive avidin-biotin ELISA method with non-modified or oxidatively modified biotinylated RuBPCO (BR) as substrates. Distinct proteolytic activities were detected. They differed in ATP and divalent metal ion dependence, protease inhibitory profile, and dynamics in the time-course of dark-induced senescence. The results supported involvement of ATP- and metal ion-dependent serine type proteolytic activity against non-modified BR early in induced senescence and appearance of ATP-independent activity at later stage. Active oxygen-modified BR was degraded by ATP-independent serine-type protease probably containing essential SH-groups and requiring divalent metal ions.     

Enzymatic activity of a synthetic 99 residue protein corresponding to the putative HIV-1 protease

A protein corresponding to the putative protease of the human immunodeficiency virus 1 (HIV-1) has been prepared by total chemical synthesis. This 99 residue synthetic enzyme showed specific proteolytic activity on fragments of the natural gag precursor and on synthetic peptide substrates, two of which released fragments corresponding to the N terminus and C terminus of the protease molecule itself. The observed substrate specificity was not restricted to cleavage at Phe/Tyr-Pro bonds. Inhibition studies provided direct evidence that the HIV-1 protease belongs to the family of aspartic proteases. The availability of the HIV-1 protease as a defined molecular species has important implications for the design of specific inhibitors that do not interfere with the host cell metabolism as a possible route to antiviral agents against acquired immunodeficiency syndrome (AIDS).     

Hepatocyte growth factor activator inhibitor type 1 inhibits protease activity and proteolytic activation of human airway trypsin-like protease

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type transmembrane serine protease inhibitor initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA), a serine protease that converts pro-HGF to the active form. HAI-1 also has inhibitory activity against serine proteases such as matriptase, hepsin and prostasin. In this study, we examined effects of HAI-1 on the protease activity and proteolytic activation of human airway trypsin-like protease (HAT), a transmembrane serine protease that is expressed mainly in bronchial epithelial cells. A soluble form of HAI-1 inhibited the protease activity of HAT in vitro. HAT was proteolytically activated in cultured mammalian cells transfected with its expression vector, and a soluble form of active HAT was released into the conditioned medium. The proteolytic activation of HAT required its own serine protease activity. Co-expression of the transmembrane full-length HAI-1 inhibited the proteolytic activation of HAT. In addition, full-length HAI-1 associated with the transmembrane full-length HAT in co-expressing cells. Like other target proteases of HAI-1, HAT converted pro-HGF to the active form in vitro. These results suggest that HAI-1 functions as a physiological regulator of HAT by inhibiting its protease activity and proteolytic activation in airway epithelium.     

Plasminogen activators and their inhibitors in the neuromuscular system: II. Serpins and serpin: protease complex receptors increase during in vitro myogenesis

In the course of studies on the regulation of plasminogen activator-mediated extracellular matrix degradation in muscle we found the presence of a factor, a cellular inhibitor of serine proteases having features similar to the serpin protease nexin I (PNI). This factor was present in the medium and at maximum concentration following fusion of skeletal muscle cells in culture. The ability of the PNI homologue in mouse muscle to inhibit ECM degradation by urokinase in myoblast medium was compared to that of human PNI purified from human fibroblasts. Stable (to SDS) 1:1 molar ratio complex formation between PNI and proteases, the proposed means by which these enzymes are regulated and removed, was also detected. Cell surface receptors for protease:PNI complexes, the specific binding sites for inactive complex internalization, were found on multinucleated myotubes, while little or no receptor activity was detected on myoblasts. These data suggest that developmental regulation of a) increased PNI proteolytic inhibitory activity expression and b) the appearance of protease:inhibitor complex receptors on muscle cell surfaces during myogenesis may constitute important regulatory features of muscle surface proteolytic activity. They complement previous studies of proteoglycan metabolism in muscle, which itself contains molecules capable of regulating the activity of myotube surface proteases.     

In vitro decondensation of the sperm chromatin in Holothuria tubulosa (sea cucumber) not affecting proteolysis of basic nuclear proteins

Sea urchin and sea star oocyte extracts contain proteolytic activities that are active against sperm basic nuclear proteins (SNBP). This SNBP degradation has been related to the decondensation of sperm chromatin as a possible model to male pronuclei formation. We have studied the presence of this proteolytic activity in Holothuria tubulosa (sea cucumber) and its possible relationship with sperm nuclei decondensation. The mature oocyte extracts from H. tubulosa contain a proteolytic activity to SNBP located in the macromolecular fraction of the egg‐jelly layer. SNBP degradation occurred both on sperm nuclei and on purified SNBP, histones being more easily degraded than protein Ø_o (sperm‐specific protein). SNBP degradation was found to be dependent on concentration, incubation time, presence of Ca²⁺, pH, and this activity could be a serine‐proteinase. Thermal denaturalization of the oocyte extracts (80°C, 10–15 min) inactivates its proteolytic activity on SNBP but does not affect sperm nuclei decondensation. These results would suggest that sperm nuclei decondensation occurs by a mechanism different from SNBP degradation. Thus, the sperm nuclei decondensation occurs by a thermostable factor(s) and the removal of linker SNBP (H1 and protein Ø_o) will be a first condition in the process of sperm chromatin remodeling.     

HDL3 degradation by proteases in isolated rat intestinal mucosal cells

Human 125I-labelled HDL3 is degraded by isolated rat intestinal mucosal cells. In our experimental conditions, lipoprotein degradation occurred by two different mechanisms. In one, lipoprotein was degraded within the cell, following binding and internalisation. In the other, degradation occurred in the medium, which seemed to contain protease activity released from cells during incubation. Though lipoprotein-deficient serum apparently interfered with degradation in the medium, bovine serum albumin had no such effect. The lysosomal inhibitor, chloroquine, reduced degradation by 60% without inhibiting HDL binding. Intestinal cell extracts contained at least two different proteases, with pH optima of 4.5 and 8.0, respectively. Comparing HDL and LDL degradation on a molar basis, more HDL particles were degraded by the cell-free extracts at pH 4.5. This degradation was activated by dithiothreitol and was inhibited by iodoacetic acid. From these observations we conclude that HDL3 is taken up by the rat intestinal mucosal cell through a specific binding site and subsequently degraded by a thiol-dependent protease in the lysosome.