Protease So from Escherichia coli preferentially degrades oxidatively damaged glutamine synthetase

After oxidative damage (e.g. induced with iron, ascorbate, and oxygen), the inactivated glutamine synthetase is selectively hydrolyzed in extracts of Escherichia coli. We therefore tested if glutamine synthetase treated with this system is hydrolyzed preferentially by any of the known E. coli proteases. Protease So, a cytoplasmic serine protease, was found to degrade the oxidized form of glutamine synthetase to acid-soluble peptides 5-10 times faster than the native glutamine synthetase. Degradation of the oxidized glutamine synthetase was inhibited by EDTA and stimulated 5-10-fold by Mg2+, Ca2+, or Mn2+, even though casein hydrolysis by protease So is not affected by divalent cations. Apparently, these cations affect the conformation of this substrate, making it more susceptible to proteolytic attack. Protease Re, another cytoplasmic protease, also degrades preferentially the oxidized form of glutamine synthetase and seems to correspond to the glutamine synthetase-degrading activity recently described by Roseman and Levine [1987) J. Biol. Chem. 262, 2101-2110). However, it is much less active in this reaction than protease So. No other soluble E. coli protease, including Do, Ci, Mi, Fa, Pi, or the ATP-dependent proteases Ti and La (the lon product), appears to degrade this oxidized protein. These results suggest that protease So participates in the hydrolysis of oxidatively damaged proteins and that E. coli has multiple systems for degrading different types of aberrant proteins.     

Purification and characterization of protease Re, a cytoplasmic endoprotease in Escherichia coli.

Protease Re, a new cytoplasmic endoprotease in Escherichia coli, was purified to homogeneity by conventional procedures, using [3H]casein as the substrate. The enzyme consists of a single polypeptide of 82,000 molecular weight. It is maximally active between pH 7 and 8.5 and is independent of ATP. It has a pI of 6.8 and a Km of 10.8 microM for casein. Since diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride inhibited this enzyme, it appears to be a serine protease. Protease Re was sensitive to inhibition by L-1-tosylamido-2-phenylethylchloromethylketone but not to that by 1-chloro-3-tosylamido-7-aminoheptanone, thiol-blocking reagents, chelating agents, or various peptide aldehydes. Re also degraded [125I]globin, [125I]glucagon, and 125I-labeled denatured bovine serum albumin to acid-soluble products (generally oligopeptides of greater than 1,500 daltons), but it showed no activity against serum albumin, growth hormone, insulin, or a variety of fluorometric peptide substrates. It also hydrolyzed oxidatively inactivated glutamine synthetase (generated by ascorbate, oxygen, and iron) four- to fivefold more rapidly than the native protein. Protease Re appears to be identical to the proteolytic enzyme isolated by Roseman and Levine (J. Biol. Chem. 262:2101-2110, 1987) by its ability to degrade selectively oxidatively damaged glutamine synthetase in vivo. Its role in intracellular protein breakdown is uncertain.     

Purification and characterization of protease So, a cytoplasmic serine protease in Escherichia coli

A new cytoplasmic endoprotease, named protease So, was purified to homogeneity from Escherichia coli by conventional procedures with casein as the substrate. Its molecular weight was 140,000 when determined by gel filtration on Sephadex G-200 and 77,000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Thus, it appears to be composed of two identical subunits. Protease So had an isoelectric point of 6.4 and a K(m) of 1.4 muM for casein. In addition to casein, it hydrolyzed globin, glucagon, and denatured bovine serum albumin to acid-soluble peptides but did not degrade insulin, native bovine serum albumin, or the "auto alpha" fragment of beta-galactosidase. A variety of commonly used peptide substrates for endoproteases were not hydrolyzed by protease So. It had a broad pH optimum of 6.5 to 8.0. This enzyme is a serine protease, since it was inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. Although it was not inhibited by chelating agents, divalent cations (e.g., Mg(2+)) stabilized its activity. Protease So was sensitive to inhibition by N-tosyl-l-phenylalanine chloromethyl ketone but not by N-tosyl-l-lysine chloromethyl ketone. Neither ATP nor 5'-diphosphate-guanosine-3'-diphosphate affected the rate of casein hydrolysis. Protease So was distinct from the other soluble endoproteases in E. coli (including proteases Do, Re, Mi, Fa, La, Ci, and Pi) in its physical and chemical properties and also differed from the membrane-associated proteases, protease IV and V, and from two amino acid esterases, originally named protease I and II. The physiological function of protease So is presently unknown.     

The Escherichia coli heat shock protease HtrA participates in defense against oxidative stress

The serine protease HtrA (DegP), which is indispensable for cell survival at elevated temperatures, is a peripheral membrane protein, localized on the periplasmic side of the inner membrane in Escherichia coli, and the biochemical and genetic evidence indicates that the physiological role of HtrA is to degrade denatured proteins formed in the cellular envelope during heat shock. The aim of this study was to find out if the HtrA protease contributes to protection of the cell against oxidative stress. We compared the influence of various oxidizing agents on htrA mutant cells with their effects on wild-type bacteria, and found that the htrA mutation did not increase sensitivity to hydrogen peroxide or paraquat but made the cell extremely sensitive to ferrous [Fe(II)] ions, which are known to enhance oxidation of proteins. Treatment with ferrous ions caused a larger increase in the level of protein carbonyl groups in the membrane fraction of the cell than in the periplasm and cytoplasm. Iron-induced oxidation of membrane proteins was enhanced in the htrA mutant relative to wild-type cells. Inhibition of the growth of the htrA mutant by iron could be alleviated more efficiently by a nitroxide antioxidant that localizes in the membranes (A-TEMPO) than by a derivative (4OH-TEMPO) that acts mainly in the soluble fraction of the cell. Inhibition of the growth of the htrA mutant was more pronounced following treatment with cumene hydroperoxide, which partitions into membranes, than with t-butyl hydroperoxide, which forms radical mainly in the cytosol. Both ferrous ions and cumene hydroperoxide, but not hydrogen peroxide, paraquat or t-butyl hydroperoxide, induced synthesis of HtrA. Our results show that HtrA plays a role in defense against oxidative shock and support the hypothesis that HtrA participates in the degradation of oxidatively damaged proteins localized in the cell envelope, especially those associated with the membranes.     

Characterization, localization and transmembrane organization of the three proteins PrtD, PrtE and PrtF necessary for protease secretion by the Gram-negative bacterium Erwinia chrysanthemi

Erwinia chrysanthemi, a Gram-negative phythopathogenic bacterium, secretes two related extracellular metalloproteases, B and C, which do not have N-terminal signal sequences. The specific pathway by which they are secreted, which has been reconstituted in Escherichia coli, comprises three proteins -- PrtD, PrtE and PrtF. Hybrid proteins containing segments of these proteins fused to the C-terminus of protease B were purified and used to immunize rabbits. The antisera thus obtained were used to study the location and membrane topology of the three proteins. PrtD and PrtE were found to cofractionate almost exclusively with the cytoplasmic membrane, whereas PrtF was found to co-fractionate mostly with the outer membrane. Proteinase K accessibility experiments as well as sequence data lead us to propose that PrtF has one or both ends exposed to the periplasm, that PrtE has one transmembrane segment with its amino-terminus facing the cytoplasm and its C-terminal hydrophilic domain exposed to the periplasm, and that PrtD has six transmembrane segments with its N-terminus and its C-terminal hydrophilic domain in the cytoplasm.     

The Escherichia coli heat shock protease HtrA participates in defense against oxidative stress

The serine protease HtrA (DegP), which is indispensable for cell survival at elevated temperatures, is a peripheral membrane protein, localized on the periplasmic side of the inner membrane in Escherichia coli, and the biochemical and genetic evidence indicates that the physiological role of HtrA is to degrade denatured proteins formed in the cellular envelope during heat shock. The aim of this study was to find out if the HtrA protease contributes to protection of the cell against oxidative stress. We compared the influence of various oxidizing agents on htrA mutant cells with their effects on wild-type bacteria, and found that the htrA mutation did not increase sensitivity to hydrogen peroxide or paraquat but made the cell extremely sensitive to ferrous [Fe(II)] ions, which are known to enhance oxidation of proteins. Treatment with ferrous ions caused a larger increase in the level of protein carbonyl groups in the membrane fraction of the cell than in the periplasm and cytoplasm. Iron-induced oxidation of membrane proteins was enhanced in the htrA mutant relative to wild-type cells. Inhibition of the growth of the htrA mutant by iron could be alleviated more efficiently by a nitroxide antioxidant that localizes in the membranes (A-TEMPO) than by a derivative (4OH-TEMPO) that acts mainly in the soluble fraction of the cell. Inhibition of the growth of the htrA mutant was more pronounced following treatment with cumene hydroperoxide, which partitions into membranes, than with t-butyl hydroperoxide, which forms radical mainly in the cytosol. Both ferrous ions and cumene hydroperoxide, but not hydrogen peroxide, paraquat or t-butyl hydroperoxide, induced synthesis of HtrA. Our results show that HtrA plays a role in defense against oxidative shock and support the hypothesis that HtrA participates in the degradation of oxidatively damaged proteins localized in the cell envelope, especially those associated with the membranes. Received: 9 March 1999 / Accepted: 31 May 1999     

Either periplasmic tethering or protease resistance is sufficient to allow a SodC to protect Salmonella enterica serovar Typhimurium from phagocytic superoxide

Salmonella Typhimurium combats phagocytic superoxide by producing the periplasmic superoxide dismutase, SodCI. The homologous protein, SodCII, is also produced during infection, but does not contribute to virulence. The proteins physically differ in that SodCI is dimeric, protease resistant and non-covalently tethered within the periplasm. Conversely, SodCII is a protease-sensitive monomer that is released normally from the periplasm by osmotic shock. To identify which properties correlate with virulence, we constructed over 20 enzymatically functional hybrid SodC proteins and assayed them for protease susceptibility, release by osmotic shock, multimerization and affinity for metal cofactors. Protease susceptibility maps to the C-terminus of SodCII, while SodCI residues 120-131 are required for tethering. A protease-resistant SodCII hybrid was able to substitute for SodCI during infection. Interestingly, a tethered but protease-sensitive SodCII hybrid was also able to confer protection. Thus, either tethering or protease resistance is sufficient for a SodC to function during infection. These results support our model that in the macrophage, the outer membrane of Salmonella is partially disrupted by antimicrobial peptides. Periplasmic proteins, including SodCII, are released and/or phagocytic proteases gain access. SodCI is both tethered within the periplasm and protease resistant, thereby surviving to detoxify superoxide.     

Determinants of translocation and folding of TreF, a trehalase of Escherichia coli

One isoform of trehalase, TreF, is present in the cytoplasm and a second, TreA, in the periplasm. To study the questions of why one enzyme is exported efficiently and the other is not and whether these proteins can fold in their nonnative cellular compartment, we fused the signal sequence of periplasmic TreA to cytoplasmic TreF. Even though this TreF construct was exported efficiently to the periplasm, it was not active. It was insoluble and degraded by the periplasmic serine protease DegP. To determine why TreF was misfolded in the periplasm, we isolated and characterized Tre(+) revertants of periplasmic TreF. To further characterize periplasmic TreF, we used a genetic selection to isolate functional TreA-TreF hybrids, which were analyzed with respect to solubility and function. These data suggested that a domain located between residues 255 and 350 of TreF is sufficient to cause folding problems in the periplasm. In contrast to TreF, periplasmic TreA could fold into the active conformation in its nonnative cellular compartment, the cytoplasm, after removal of its signal sequence.     

The HtrA family of serine proteases

HtrA, also known as DegP and probably identical to the Do protease, is a heat shock-induced serine protease that is active in the periplasm of Escherichia coli . Homologues of HtrA have been described in a wide range of bacteria and in eukaryotes. Its chief role is to degrade misfolded proteins in the periplasm. Substrate recognition probably involves the recently described PDZ domains in the C-terminal half of HtrA and, we suspect, has much in common with the substrate recognition system of the tail-specific protease, Prc (which also possesses a PDZ domain). The expression of htrA is regulated by a complex set of signal transduction pathways, which includes an alternative sigma factor, RpoE, an anti-sigma factor, RseA, a two-component regulatory system, CpxRA, and two phosphoprotein phosphatases, PrpA and PrpB. Mutations in the htrA genes of Salmonella , Brucella and Yersinia cause decreased survival in mice and/or macrophages, and htrA mutants can act as vaccines, as cloning hosts and as carriers of heterologous antigens.     

Human red blood cell insulin-degrading enzyme and rat skeletal muscle insulin protease share antigenic sites and generate identical products from insulin.

The mechanisms of cellular insulin degradation remain uncertain. Considerable evidence now exists that the primary cellular insulin-degrading activity is a metallothiol proteinase. Two similar degrading activities have been purified and characterized. Insulin protease has been purified from rat skeletal muscle and insulin-degrading enzyme from human red blood cells. Whereas the two degrading activities share a number of similar properties, significant differences have also been reported; and it is not at all established that they are the same enzyme. To examine this, we have compared antigenic and catalytic properties of the two enzymatic activities. Monoclonal antibodies against the red blood cell enzyme adsorb the skeletal muscle enzyme; and on Western blots, the antibodies react with an identical 110-kDa protein. Immunoaffinity-purified enzymes from both red blood cells and skeletal muscle degrade [125I]iodo(B26)insulin to the same products as seen with purified insulin protease and with intact liver and kidney. Chelator-treated muscle and red blood cell enzymes can be reactivated with either Mn2+ or Ca2+. Thus, insulin-degrading enzyme and insulin protease have similar properties. These results support the hypothesis that these activities reside in the same enzyme.     

Degradation of the Twin-Arginine Translocation Substrate YwbN by Extracytoplasmic Proteases of Bacillus subtilis

Bacterial twin-arginine translocases can export fully folded proteins from the cytoplasm. Such proteins are usually resistant to proteolysis. Here we show that multiple extracellular proteases degrade the B. subtilis Tat substrate YwbN. This suggests either that secreted YwbN is not fully folded or that folded YwbN exposes protease cleavage sites.     

Molecular aspects of complement-mediated bacterial killing. Periplasmic conversion of C9 from a protoxin to a toxin

As part of the membrane attack complex complement protein C9 is responsible for direct killing of bacteria. Here we show that in the periplasmic space of an Escherichia coli cell C9 is converted from a protoxin to a toxin by periplasmic conditions missing in spheroplasts. This conversion is independent of the pathway by which C9 enters the periplasm. Both, C9 shocked into the periplasm and plasmid-expressed C9 targeted to the periplasm via a signal sequence are toxic. Toxicity requires disulfide-linked C9 because export into the periplasm of cells defective in disulfide bond synthesis (dsbA and dsbB mutants) is not toxic unless N-acetylcysteine is added externally to promote cystines. A N-terminal fragment, C9[1-144], is not toxic nor is cytoplasmically expressed C9, even in trxB mutants that are able to form disulfide bonds in the cytoplasm. Importantly, expression of full-length C9 in complement-resistant cells has no effect on their viability. Expression and translocation into the periplasm may provide a novel model to identify molecular mechanisms of other bactericidal disulfide-linked proteins and to investigate the nature of bacterial complement resistance.     

Transport of ferric-aerobactin into the periplasm and cytoplasm of Escherichia coli K12: role of envelope-associated proteins and effect of endogenous siderophores.

Purified [14C]aerobactin, supplied exogenously to non-growing bacteria, was translocated via the periplasm into the cytoplasm of Escherichia coli K12 strains expressing the aerobactin receptor protein IutA. No significant uptake was observed into either compartment of strains lacking the iutA gene or specifically defective in tonB. Uptake into both compartments was markedly reduced, but not abolished, in an exb mutant. Accumulation of [14C]aerobactin in the periplasm of fhuD, fhuB or fhuC mutant strains was not significantly lower than in the wild-type strain, but entry into the cytoplasm was greatly reduced in all cases. Uptake of aerobactin by strains wild-type for all transport functions occurred most efficiently in strains either lacking or specifically defective in the genetic determinants for aerobactin biosynthesis; significantly lower levels of exogenous 14C-labelled siderophore were observed in both compartments of strains producing aerobactin. Aerobactin-mediated 59Fe uptake, however, was not inhibited by the presence of endogenous aerobactin. Endogenous enterochelin did not affect aerobactin uptake.     

Escherichia coli alkaline phosphatase localized to the cytoplasm slowly acquires enzymatic activity in cells whose growth has been suspended: a caution for gene fusion studies.

Alkaline phosphatase is normally localized to the periplasm of Escherichia coli and is unable to fold into its native conformation if retained in the cytoplasm of growing cells. The alkaline phosphatase activity of E. coli expressing a version of the protein without a signal sequence was nonetheless found to increase gradually when the growth of cells was suspended. At least 30% of the protein was activated over the course of several hours when freshly grown exponential-phase cells were held on ice. Similar behavior was observed with cells expressing certain other mutant versions of alkaline phosphatase that are retained in the cytoplasm. The activation resulted not from the passage of the alkaline phosphatase into the periplasm but from the slow folding of alkaline phosphatase into its native conformation in the cytoplasm. These findings indicate that the mechanism by which proteins are normally kept reduced in the cytoplasm fails to function if cells are not growing. It was found that the addition of the sulfhydryl-alkylating agent iodoacetamide to cells after growth blocks this activation completely. This treatment can therefore diminish the likelihood of spurious enzyme activity measurements in studies that make use of alkaline phosphatase fusion proteins.     

Proteases of nuclei of testicular cells assayed with fluorogenic peptides

Nuclei of seminiferous epithelial cells (SEC) of rat testis exhibit protease activity when assayed with fluorogenic peptides or with [3H]histones. At pH 8 the nuclear protease rapidly hydrolyzes BOC-Val-Pro-Arg-7-Amino-4-Methyl Coumarin (BVPAC) and Glu-Gly-Arg-7-Amino-4-Methyl Coumarin (GGAC), but this enzyme does not hydrolyze CBZ-Arg-7-Amino-4-Methyl Coumarin (CAC) or Glu-Phe-7-Amino-4-Methyl Coumarin (GPC). The cytoplasm of these cells hydrolyzes each of these substrates. The protease activity versus BVPAC can be extracted from cytoplasm and nuclei with 0.1 M H2SO4. The extracted activity from cytoplasm is lost during storage for 5 days at either pH 3 or pH 8 at -25 degrees, but the activity extracted from nuclei is maintained under these conditions. The nuclear protease activity is found in SEC of young rats prior to the appearance of acrosin.     

Degradation in vitro of bacteriophage lambda N protein by Lon protease from Escherichia coli

Lon protease from Escherichia coli degraded lambda N protein in a reaction mixture consisting of the two homogeneous proteins, ATP, and MgCl2 in 50 mM Tris, Ph 8.0. Genetic and biochemical data had previously indicated that N protein is a substrate for Lon protease in vivo (Gottesman, S., Gottesman, M., Shaw, J. E., and Pearson, M. L. (1981) Cell 24, 225-233). Under conditions used for N protein degradation, several lambda and E. coli proteins, including native proteins, oxidatively modified proteins, and cloned fragments of native proteins, were not degraded by Lon protease. Degradation of N protein occurred with catalytic amounts of Lon protease and required the presence of ATP or an analog of ATP. This is the first demonstration of the selective degradation of a physiological substrate by Lon protease in vitro. The turnover number for N protein degradation was approximately 60 +/- 10 min-1 at pH 8.0 in 50 mM Tris/HCl, 25 mM MgCl2 and 4 mM ATP. By comparison the turnover number for oxidized insulin B chain was 20 min-1 under these conditions. Kinetic studies suggest that N protein (S0.5 = 13 +/- 5 microM) is intermediate between oxidized insulin B chain (S0.5 = 160 +/- 10 microM) and methylated casein (S0.5 = 2.5 +/- 1 microM) in affinity for Lon protease. N protein was extensively degraded by Lon protease with an average of approximately six bonds cleaved per molecule. In N protein, as well as in oxidized insulin B chain and glucagon, Lon protease preferentially cut at bonds at which the carboxy group was contributed by an amino acid with an aliphatic side chain (leucine or alanine). However, not all such bonds of the substrates were cleaved, indicating that sequence or conformational determinants beyond the cleavage site affect the ability of Lon protease to degrade a protein.     

绿木霉Gv29-8丝氨酸蛋白酶S8/S53超家族基因特性及功能

【背景】丝氨酸蛋白酶在木霉菌生物防治过程中发挥重要作用。【目的】研究绿木霉丝氨酸蛋白酶S8/S53超家族基因信息及其生物学功能,进而为该蛋白酶生防制剂的开发及基因改造提供理论支持。【方法】通过生物信息学分析方法,从绿木霉Gv29-8基因组中鉴定出23个丝氨酸蛋白酶基因,以少孢节丛孢菌ATCC 24927基因组中鉴定的4个丝氨酸蛋白酶基因作为对照,对这27个丝氨酸蛋白酶基因的特性、蛋白结构、进化地位、功能等进行预测分析。【结果】27个基因结构差异较大,编码的蛋白具有典型的丝氨酸蛋白酶催化三联体结构,属于S8/S53超家族,分为6个亚家族,同一亚家族的蛋白酶保守区长度相近,相似性较高,催化残基附近序列比较保守。系统进化分析显示,同一亚家族丝氨酸蛋白酶聚为一类。【结论】绿木霉和少孢节丛孢菌的部分丝氨酸蛋白酶基因在结构和蛋白性质上相似性强,亲缘关系较近,均属于S8_PCSK9_ProteinaseK_like亚家族,推测绿木霉与少孢节丛孢菌该亚家族的丝氨酸蛋白酶具有相似的功能,可抑制植物病原真菌和降解线虫体壁。     

Efficient export of prefolded,disulfide‐bonded recombinant proteins to the periplasm by the Tat pathway in Escherichia coli CyDisCo strains

Numerous high‐value therapeutic proteins are produced in Escherichia coli and exported to the periplasm, as this approach simplifies downstream processing and enables disulfide bond formation. Most recombinant proteins are exported by the Sec pathway, which transports substrates across the plasma membrane in an unfolded state. The Tat system also exports proteins to the periplasm, but transports them in a folded state. This system has attracted interest because of its tendency to transport correctly folded proteins, but this trait renders it unable to export proteins containing disulfide bonds since these are normally acquired only in the periplasm; reduced substrates tend to be recognized as incorrectly folded and rejected. In this study we have used a series of novel strains (termed CyDisCo) which oxidise disulfide bonds in the cytoplasm, and we show that these cells efficiently export a range of disulfide‐containing proteins when a Tat signal peptide is attached. These test proteins include alkaline phosphatase (PhoA), a phytase containing four disulfide bonds (AppA), an antiinterleukin 1β scFv and human growth hormone. No export of PhoA or AppA is observed in wild‐type cells lacking the CyDisCo factors. The PhoA, AppA and scFv proteins were exported in an active form by Tat in the CyDisCo strain, and mass spectrometry showed that the vast majority of the scFv protein was disulfide‐bonded and correctly processed. The evidence indicates that this combination of Tat + CyDisCo offers a novel means of exporting active, correctly folded disulfide bonded proteins to the periplasm. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:281–290, 2014