Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki

Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.     

Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki.

Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.     

Secretion processing and activation of Erwinia chrysanthemi proteases

E chrysanthemi, a phytopathogenic enterobacterium, secretes several enzymes into the medium such as pectinases cellulases and proteases. It also produces 3 distinct and antigenically related extracellular proteases. The proteases secretion pathway seems to be distinct from that of the other extracellular enzymes since pleiotropic mutants impaired in cellulase and pectinase secretion are unimpaired in protease secretion. E chrysanthemi proteases B and C secretion occurs without an N-terminal signal peptide and is dependent upon specific secretion functions which are encoded by genes adjacent to the protease structural genes. This secretion pathway might be analogous to the alpha-hemolysin secretion pathway in E coli. Protection against intracellular proteolytic activity is achieved by 2 distinct mechanisms: the proteases are synthesized as inactive precursors with an N-terminal extension of 15 aminoacids (protease B) and 17 aminoacids (protease C) absent in the mature active extracellular enzymes; an intracellular specific protease inhibitor is produced by some E chrysanthemi strains.     

Increased proteolytic susceptibility of carboxypeptidase Y caused by modification of the disulfide zipper

To investigate the structural importance of a "disulfide zipper" motif of carboxypeptidase Y, disulfide-deficient mutant enzymes were expressed in two strains of Saccharomyces cerevisiae. The mutant enzymes were rapidly degraded into fragments by intracellular proteases. Thus, it is concluded that the disulfide zipper is essential in maintaining the structural integrity of CPase Y against proteolytic susceptibility.     

Characterization of Erwinia chrysanthemi extracellular proteases: cloning and expression of the protease genes in Escherichia coli.

Erwinia chrysanthemi, a phytopathogenic enterobacterium, secretes three antigenically and structurally distinct proteases, A, B, and C and produces a protease inhibitor, a low-molecular-weight, heat-stable protein which remains mostly intracellular and which binds specifically to the A, B, and C proteases. The structural genes for proteases A, B, and C and for the inhibitor are clustered on a ca. 40-kilobase DNA fragment present in cosmid pEW4. Escherichia coli strains harboring pEW4 secrete the three proteases into the medium during the exponential phase of growth, without intracellular accumulation and in the absence of detectable cell lysis. An 8.5-kilobase EcoRI fragment derived from the cosmid encodes proteases B and C and the inhibitor as well as functions involved in the synthesis or secretion (or both) of the proteases. The inhibitor is not required for protease synthesis or secretion.     

The subtilisin-like protease AprV2 is required for virulence and uses a novel disulphide-tethered exosite to bind substrates

Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have been postulated to play a role in virulence. We have constructed protease mutants of D. nodosus; their analysis in a sheep virulence model revealed that one of these enzymes, AprV2, was required for virulence. These studies challenge the previous hypothesis that the elastase activity of AprV2 is important for disease progression, since aprV2 mutants were virulent when complemented with aprB2, which encodes a variant that has impaired elastase activity. We have determined the crystal structures of both AprV2 and AprB2 and characterized the biological activity of these enzymes. These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions. The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important. Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism. In conclusion, we have used an integrated multidisciplinary combination of bacterial genetics, whole animal virulence trials in the original host, biochemical studies, and comprehensive analysis of crystal structures to provide the first definitive evidence that the extracellular secreted proteases produced by D. nodosus are required for virulence and to elucidate the molecular mechanism by which these proteases bind to their natural substrates. We postulate that this exosite mechanism may be used by proteases produced by other bacterial pathogens of both humans and animals.     

Selection and characterization of a proteo-chitinolytic strain of Bacillus thuringiensis,able to grow in shrimp waste media

This paper reports the selection and characterization of Bacillus thuringiensis strains, with ability to grow in a proteo-chitinaceous substrate (milled shrimp waste) as the sole ingredient. Selected strains were able to produce crystal proteins, as well as proteases and chitinases as fermentation by-products. By a preliminary, qualitative screening of 152 B. thuringiensis strains, grown on media rich in protein and chitin, eight strains were selected. These strains were cultured in a liquid medium containing milled shrimp waste and their kinetics of protease production were followed. The two most active proteolytic strains (Bt-103 and Bt-112) were characterized by their crystal protein content, plasmid profiles, crystal ultrastructure, and toxicity towards Manduca sexta, Aedes aegypti and Leptinotarsa texana. The only activity recorded in these species was moderate toxicity of strain Bt-112 against Manduca sexta first instar larvae, as well as the highest proteolytic and chitinolytic activities. Its bipyramidal crystals were associated with semi-cuboidal inclusions and although its crystal proteins were similar to those of B. thuringiensis kurstaki (HD-1), its plasmid content was quite different. Serotyping of Bt-112 indicated that it belongs to serovar. tolworthi. Further studies with a similar strategy might render more strains with ability to grow in a rich waste by-product like the shrimp waste, which may show not only higher insecticidal activity, but also with the ability to produce extracellular enzymes with biotechnological applications.     

Production by fungi of the genera Aspergillus, Acremonium, Verticillium of extracellular proteases which coagulate blood plasma and lyse blood clots

Among 24 fungal strains belonging to the genera Aspergillus, Acremonium and Verticillium, eight strains synthesized proteases with the coagulating activity. The most active strains producing such proteases were found among Aspergillus species. The fibrinolytic activity was detected in 12 strains of the fungi. Proteases with the fibrinolytic activity differed in their sensitivity to plasma inhibitors and the least sensitive proteases were found in A. niger and A. flavus. Some fungal strains synthesized proteases with the fibrinolytic activity exerting an indirect action. Such enzymes activated the conversion of blood profibrinolysin into fibrinolysin in a manner similar to that of staphylokinase, urokinase and trypsin.     

Structural basis for c-di-GMP-mediated inside-out signaling controlling periplasmic proteolysis

The bacterial second messenger bis-(3'-5') cyclic dimeric guanosine monophosphate (c-di-GMP) has emerged as a central regulator for biofilm formation. Increased cellular c-di-GMP levels lead to stable cell attachment, which in Pseudomonas fluorescens requires the transmembrane receptor LapD. LapD exhibits a conserved and widely used modular architecture containing a HAMP domain and degenerate diguanylate cyclase and phosphodiesterase domains. c-di-GMP binding to the LapD degenerate phosphodiesterase domain is communicated via the HAMP relay to the periplasmic domain, triggering sequestration of the protease LapG, thus preventing cleavage of the surface adhesin LapA. Here, we elucidate the molecular mechanism of autoinhibition and activation of LapD based on structure-function analyses and crystal structures of the entire periplasmic domain and the intracellular signaling unit in two different states. In the absence of c-di-GMP, the intracellular module assumes an inactive conformation. Binding of c-di-GMP to the phosphodiesterase domain disrupts the inactive state, permitting the formation of a trans-subunit dimer interface between adjacent phosphodiesterase domains via interactions conserved in c-di-GMP-degrading enzymes. Efficient mechanical coupling of the conformational changes across the membrane is realized through an extensively domain-swapped, unique periplasmic fold. Our structural and functional analyses identified a conserved system for the regulation of periplasmic proteases in a wide variety of bacteria, including many free-living and pathogenic species.     

Tempe fermentation: some aspects of formation of gamma-linolenic acid, proteases and vitamins

During a tempe fermentation the concentrations of linoleic, and alpha-linolenic acids (ALA) decreased while the concentration of oleic acid increased. During fatty acid synthesis Rhizopus sp. produced only gamma-linolenic acid (GLA) instead of ALA. The amount of GLA in tempe were influenced by varying external parameters.The proteolytic capacity of 36 strains of the genus Rhizopus isolated from Indonesian tempe or tempe inocula was examined. There was a distinct increase in the amount of free amino acids during tempe fermentation. Fermentations with mixed populations of bacteria and Rhizopus yielded a lower level of free amino acids, but an increase in total amount of amino acids. In comparison to intracellular, and extracellular proteases the proteases of the cell wall fraction are most responsible for proteolytic capacity of the different Rhizopus strains.Two isolated strains of Citrobacter freundii were found to be the best vitamin B(12) producers during the soaking of soybeans. In the solid substrate fermentation the Rhizopus molds formed vitamin B(6), riboflavin, and nicotinic acid. The addition of bacteria to the solid substrate fermentation resulted in a strong increase of active vitamin B(12) in tempe. In the presence of the Rhizopus mold, the vitamin B(12) formation by C. freundii was three times higher than that of a fermentation without the mold.     

Activities and partial purification of extracellular proteases of Bacteroides nodosus from virulent and benign footrot

In an attempt to differentiate virulent and benign strains of B. nodosus, the extracellular proteolytic activity of these cultures was assayed with elastin, casein and hide powder azure, and the stability to heating at 55 degrees C was determined. Broth cultures of both strains hydrolysed 125I-labelled elastin, indicating that this activity is not a unique marker of virulence. When cultures were grown in Trypticase-arginine-serine broth medium modified by omitting Na2CO3 and thioglycollic acid, the total proteolytic activity and its stability at 55 degrees C could be used to differentiate isolates causing virulent or benign footrot lesions. However, when other broth cultures were used, these parameters could no longer be used to make such a distinction. The proteases of a virulent and benign strain of B. nodosus were partially purified and characterized. Four to five closely related proteases were detected by polyacrylamide gel electrophoresis at pH 8.8 in both types of isolates. The proteases are serine-type enzymes requiring a divalent metal ion such as calcium for activity. The proteases of the benign strain were somewhat less stable to heat than the enzymes of the virulent strain. Differences in the relative mobilities of the proteases of virulent and benign strains of B. nodosus, on electrophoresis at pH 8.8, suggest that this property may be used to distinguish virulent and benign strains.     

The vacuolar H+ -ATPase mediates intracellular acidification required for neurodegeneration in C. elegans

Numerous studies implicate necrotic cell death in devastating human pathologies such as stroke and neurodegenerative diseases. Investigations in both nematodes and mammals converge to implicate specific calpain and aspartyl proteases in the execution of necrotic cell death. It is believed that these proteases become activated under conditions that inflict necrotic cell death. However, the factors that modulate necrosis and govern the erroneous activation of these otherwise benign enzymes are largely unknown. Here we show that the function of the vacuolar H(+)-ATPase, a pump that acidifies lysosomes and other intracellular organelles, is essential for necrotic cell death in C. elegans. Cytoplasmic pH drops in dying cells. Intracellular acidification requires the vacuolar H(+)-ATPase, whereas alkalization of endosomal and lysosomal compartments by weak bases protects against necrosis. In addition, we show that vacuolar H(+)-ATPase activity is required downstream of cytoplasmic calcium overload during necrosis. Thus, intracellular pH is an important modulator of necrosis in C. elegans. We propose that vacuolar H(+)-ATPase activity is required to establish necrosis-promoting, acidic intracellular conditions that augment the function of executioner aspartyl proteases in dying cells. Similar mechanisms may contribute to necrotic cell death that follows extreme acidosis-for example, during stroke-in humans.     

Characterization of alanyl aminopeptidase from insecticide resistant and susceptible strains of Musca domestica L.

To investigate the high activity of intracellular proteases in insecticide resistant strains of Musca domestica L., purification by anion‐exchange chromatography and gel filtration of one of the enzymes, alanyl aminopeptidase (Ala AP), in three strains of Musca domestica was carried out. The fractions collected by gel filtration of soluble homogenates of the three strains (571ab, 17bb and Cooper) showed a single peak of Ala AP activity. Partially purified Ala AP of the three strains showed high activity at pH 7.5. The presence or absence of Ca²⁺ in the assay medium did not produce any difference in activity of Ala AP in the 571ab and Cooper strains, but there was a significant difference in the 17bb strain. The activity of Ala AP in all three strains was essentially unaltered in the presence of inhibitors of serine (PMSF), cysteine (E‐64) proteases and carboxypeptidases (pepstatin). Ala AP hydrolyzed alanine amino methylcoumarin (Ala‐AMC) maximally, followed by phenyl alanine amino methylcoumarin (Phe‐AMC), leucyl amino methylcoumarin (Leu‐AMC) and ornithine amino methylcoumarin (Orn‐AMC). Ala AP from the three strains showed differential activity towards various substrates. The comparison of alanyl aminopeptidase's activity from different sources is discussed.     

Intracellular proteolytic systems may function as secondary antioxidant defenses: An hypothesis

In recent years it has become clear that various free radicals and related oxidants can cause serious damage to intracellular enzymes and other proteins. Several investigators have shown that in extreme cases this can result in an accumulation of oxidatively damaged proteins as useless cellular debris. In other instances, proteins may undergo scission reactions with certain radicals/oxidants, resulting in the direct formation of potentially toxic peptide fragments. Data has also been gathered (recently) demonstrating that various intracellular proteolytic enzymes or systems can recognize, and preferentially degrade, oxidatively damaged proteins (to amino acids). In this hypothesis paper I present evidence to suggest that proteolytic systems (of proteinases, proteases, and peptidases) may function to prevent the formation or accumulation of oxidatively damaged protein aggregates. Proteolytic systems can also preferentially degrade peptide fragments and may thus prevent a wide variety of potentially toxic consequences. I propose that many proteolytic enzymes may be important components of overall antioxidant defenses because they can act to ameliorate the consequences of oxidative damage. A modified terminology is suggested in which the primary antioxidants are such agents as vitamin E, β-carotene, and uric acid and such enzymes as Superoxide dismutase, glutathione peroxidase, and DT-diaphorase. In this classification scheme, proteolytic systems, DNA repair systems, and certain lipolytic enzymes would be considered as secondary antioxidant defenses. As secondary antioxidant defenses, proteolytic systems may be particularly important in times of high oxidative stress, during periods of (primary) antioxidant insufficiency, or with advancing age.     

Elevated levels of Ca(II) modulate the activity and inhibition of serine proteases: implication in the mechanism of apoptosis

Elevated levels of intracellular Ca(II) are a prominent feature of apoptosis, a natural form of cell death involved in many physiological and pathological processes. Serine proteases play crucial roles in apoptosis and have been implicated in the genomic DNA degradation and the massive protein degradation that occur during apoptosis. In this study, the effects of the elevated level of Ca(II) on the activity and inhibition of serine proteases were examined by spectrophotometric methods. The effects of the elevated levels of Ca(II), Mg(II), K(I), and Na(I) on the activity and inactivation of three representative members of serine proteases were determined. The level of serine protease activity in CEM-C7-14 leukemic cells was also evaluated in the presence and absence of dexamethasone-induced apoptosis, and also in the presence of A23187, a Ca(II)-ionophore. Among the four metal-ions studied, only Ca(II) was found to significantly enhance the activity of mammalian serine proteases. Ca(II) was also found to significantly protect the enzymes from inhibition, while the other three metal-ions showed no significant effect on the inactivation of the enzymes. Compared to the control sample, the enzymic activity was found to be higher during apoptosis, and in the presence of the Ca(II)-ionophore. Results of this study indicate that Ca(II) can significantly enhance the catalytic efficiency of serine proteases during apoptosis.     

Intracellular protease activity during the growth of Streptomyces coelicolor

Multiple intracellular proteases were produced by Streptomyces coelicolor throughout growth as surface cultures. Zymography revealed two constitutive, gelatinolytic proteases of approximate molecular masses 32.5 and 36.5 kDa. In addition, transient expression of a large (183.5 kDa) protease preceded aerial mycelium formation and following this, during sporulation, an additional protease of mass 27.5 kDa was produced.     

Protease secretion in Neurospora crassa

Secreted and constitutive intracellular proteases of $\text{Neurospora crassa}$ differ with regard to inhibitor sensitivity, substrate specificity, isoelectric points and other properties. Upon the induction of protease secretion the enzymes released from the mycelium are formed $\text{de novo}$ as demonstrated by density labelling with D₂O. Vesicles which contain the constitutive intracellular proteases are, therefore, not involved in the secretion of proteases.     

Proteinases and exopeptidases from the phytopathogenic fungus Ustilago maydis

The proteolytic system of the phytopathogenic and dimorphic fungus Ustilago maydis is not known. In this work, we report the presence of at least four proteases from two haploid strains of U. maydis. Activities of two proteinases pumA and pumB, aminopeptidase pumAPE, and dipeptidylaminopeptidase pumDAP were measured under several nutritional and morphological conditions, including the yeast-mycelium transition. The activity of pumA was found in the intracellular and extracellular fractions, pumAi and pumAe, respectively. The latter activity was detected only during the yeast-mycelium dimorphic transition induced by growth at acid pH in a medium containing ammonium as the sole nitrogen source. Activity of pumAe was partially inhibited by Pepstatin A, which also inhibited mycelium formation. Activity of pumAi was inhibited by this specific inhibitor of aspartyl-proteases. Activity of pumB was detected in intracellular and extracellular fractions, mostly bound to an endogenous inhibitor, which was removed by treatment at acid pH. This fungus contains at least two soluble pumAPE, which might be metallo-proteases, because they were inhibited by EDTA and 1-10, phenanthroline. When the fungus was grown in media containing proline or corn infusion as the nitrogen source, an intracellular pumDAP activity was detected. No carboxypeptidase activity was found with N-benzoyl-l-tyrosine-4-nitroanilide as substrate in any of the conditions tested in any of the U. maydis strains analyzed.     

Implications of protease activation in cardiac dysfunction and development of genetic cardiomyopathy in hamsters

It has become evident that protein degradation by proteolytic enzymes, known as proteases, is partly responsible for cardiovascular dysfunction in various types of heart disease. Both extracellular and intracellular alterations in proteolytic activities are invariably seen in heart failure associated with hypertrophic cardiomyopathy, dilated cardiomyopathy, hypertensive cardiomyopathy, diabetic cardiomyopathy, and ischemic cardiomyopathy. Genetic cardiomyopathy displayed in different strains of hamsters provides a useful model for studying heart failure due to either cardiac hypertrophy or cardiac dilation. Alterations in the function of several myocardial organelles such as sarcolemma, sarcoplasmic reticulum, myofibrils, mitochondria, as well as extracellular matrix have been shown to be due to subcellular remodeling as a consequence of changes in gene expression and protein content in failing hearts from cardiomyopathic hamsters. In view of the increased activities of various proteases, including calpains and matrix metalloproteinases in the hearts of genetically determined hamsters, it is proposed that the activation of different proteases may also represent an important determinant of subcellular remodeling and cardiac dysfunction associated with genetic cardiomyopathy.     

Clp-dependent proteolysis down-regulates central metabolic pathways in glucose-starved Bacillus subtilis

Entry into stationary phase in Bacillus subtilis is linked not only to a redirection of the gene expression program but also to posttranslational events such as protein degradation. Using ³⁵S-labeled methionine pulse-chase labeling and two-dimensional polyacrylamide gel electrophoresis we monitored the intracellular proteolysis pattern during glucose starvation. Approximately 200 protein spots diminished in the wild-type cells during an 8-h time course. The degradation rate of at least 80 proteins was significantly reduced in clpP, clpC, and clpX mutant strains. Enzymes of amino acid and nucleotide metabolism were overrepresented among these Clp substrate candidates. Notably, several first-committed-step enzymes for biosynthesis of aromatic and branched-chain amino acids, cell wall precursors, purines, and pyrimidines appeared as putative Clp substrates. Radioimmunoprecipitation demonstrated GlmS, IlvB, PurF, and PyrB to be novel ClpCP targets. Our data imply that Clp proteases down-regulate central metabolic pathways upon entry into a nongrowing state and thus contribute to the adaptation to nutrient starvation. Proteins that are obviously nonfunctional, unprotected, or even “unemployed” seem to be recognized and proteolyzed by Clp proteases when the resources for growth become limited.