Degradation of proline peptides in peptidase-deficient strains of Salmonella typhimurium.

A mutant strain of Salmonella typhimurium that lacks two proline-specific peptidases (peptidases P and Q) could not complete the degradation of proline peptides formed as intermediates in starvation-induced protein breakdown. The wild-type strain produced free proline as the product of degradation of proline-labeled proteins. The pepP pepQ mutant, however, produced a mixture of small proline peptides. In the absence of peptidase Q only, peptidase P could complete the degradation of most of the proline peptide intermediates formed. In the absence of peptidase P only, about 50% of the proline-labeled, acid-soluble products were proline peptides. These results are consistent with in vitro specificity data indicating that peptidase Q hydrolyzes X-Pro dipeptides only, whereas peptidase P attacks both X-Pro dipeptides and longer peptides with X-Pro at their N-termini. A mutant strain lacking four broad-specificity peptidases (peptidases N, A, B, and D), but containing peptidases P and Q, also produced proline peptides as products of protein breakdown. This observation suggests that broad-specificity peptidases are required to generate the X-Pro substrates of peptidases P and Q. A strain lacking six peptidases (N, A, B, D, P, and Q) was constructed and produced less free proline from protein breakdown than either the pepP pepQ strain or the pepN pepA pepB pepD strain. These observations suggest that the degradation of peptide intermediates involves the sequential removal of N-terminal amino acids and requires both broad-specificity aminopeptidases (peptidases N, A, and B) and the X-Pro-specific aminopeptidase, peptidase P.     

Degradation of intracellular protein in Salmonella typhimurium peptidase mutants

Multiply peptidase-deficient mutant strains of Salmonella typhimurium fail to carry out normal protein degradation during starvation for a carbon source. In these mutants, the extent of protein breakdown during starvation is about fourfold less than in the wild type. The products of protein breakdown in the mutant are mainly small, trichloroacetic acid-soluble peptides, not free amino acids as in the wild type. The carbon-starved mutant strain produces only about one thirtieth as much free amino acid from protein as the wild type. As a result, protein synthesis during starvation is reduced in the mutant compared to the wild type and the mutant strain shows a greatly prolonged lag phase after a nutritional shift-down.     

Peptide accumulation during growth of peptidase deficient mutants

Mutant strains of Salmonella typhimurium simultaneously lacking peptidases N, A, B and D accumulate a heterogeneous mixture of small, trichloroacetic acid-soluble peptides during growth in minimal medium. Approximately 20% of the labelled leucine supplied to a growing culture of the mutant strain is converted to peptides. These peptides accumulate inside the cells before being released into the growth medium. Although the origin of these peptides has not been established, there are several processes that might contribute peptides to this pool. These include (1) turnover of signal sequences, (2) turnover of attenuator peptides, and (3) degradation of prematurely terminated proteins. These results indicate that the same family of peptidases that catabolizes exogenously supplied peptides and functions in carbon-starvation-induced protein turnover also hydrolyzes peptides generated during normal exponential growth.     

Degradation of Escherichia coli beta-galactosidase fragments in protease-deficient mutants of Salmonella typhimurium.

The degradation rates of several mutationally generated fragments of Escherichia coli beta-galactosidase were determined in wild-type strains of Salmonella typhimurium and in mutant Salmonella strains lacking several proteases and peptidases. Three termination fragments (produced by lacZ545, lacZ521, and lacZX90) and one internal reinitiation (restart) fragment [lacZpi(1)] are degraded in wild-type Salmonella strains at the same rates observed in wild-type Escherichia coli strains. Mutations that lead to loss of peptidases N, A, B, P, and Q or to loss of protease I or II do not affect the decay rates of any of these fragments. In addition, all of these peptidases and proteases are present in E coli mutants carrying deg mutations (deg mutations are known to stabilize beta-galactosidase fragments). Apparently, none of the proteases and peptidases that are currently accessible to direct genetic analysis plays a role in the early steps of the degradation of protein fragments.     

Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85

We have shown that covalent conjugation of ubiquitin to proteins is temperature-sensitive in the mouse cell cycle mutant ts85 due to a specifically thermolabile ubiquitin-activating enzyme (accompanying paper). We show here that degradation of short-lived proteins is also temperature sensitive in ts85 , in contrast to wild-type and revertant cells. While more than 70% of the prelabeled abnormal proteins (containing amino acid analogs) or puromycyl peptides are degraded within 4 hr at the permissive temperature in both ts85 and wild-type cells, less than 15% are degraded in ts85 cells at the nonpermissive temperature. Degradation of abnormal proteins and puromycyl peptides in both ts85 cells and wild-type cells is nonlysosomal and ATP-dependent. Immunochemical analysis shows a strong and specific reduction in the levels of in vivo labeled ubiquitin-protein conjugates at the nonpermissive temperature in ts85 cells. Degradation of normal, short-lived proteins is also specifically temperature sensitive in ts85 . We suggest that the contribution of ubiquitin-independent pathways to the degradation of short-lived proteins in this higher eucaryotic cell is no more than 10%, and possibly less.     

Peptidase-deficient mutants of Escherichia coli.

Mutant derivatives of Escherichia coli K-12 deficient in several peptidases have been obtained. Mutants lacking a naphthylamidase, peptidase N, were isolated by screening for colonies unable to hydrolyze L-alanine beta-naphthylamide. Other mutants were isolated using positive selections for resistance to valine peptides. Mutants lacking peptidase A, a broad-specificity aminopeptidase, were obtained by selection for resistance to L-valyl-L-leucine amide. Mutants lacking a dipeptidase, peptidase D, were isolated from a pepN pepA strain by selection for resistance to L-valyl-glycine. Starting with a pepN pepA pepD strain, selection for resistance to L-valyl-glycyl-glycine or several other valine peptides produced mutants deficient in another aminopeptidase, peptidase B. Mutants resistant to L-valyl-L-proline lack peptidase Q, an activity capable of rapid hydrolysis of X-proline dipeptides. Using these selection procedures, a strain (CM89) lacking five different peptidases has been isolated. Although still sensitive to valine, this strain is resistant to a variety of valine di- and tripeptides. The ability of this strain to use peptides as sources of amino acids is much more restricted than that of wild-type E. coli strains. Strains containing only one of the five peptidases missing in CM89 have been constructed by transduction. The peptide utilization profiles of these strains show that each of the five peptidases can function during growth in the catabolism of peptides.     

Multiple-peptidase mutants of Lactococcus lactis are severely impaired in their ability to grow in milk.

To examine the contribution of peptidases to the growth of lactococcus lactis in milk, 16 single- and multiple-deletion mutants were constructed. In successive rounds of chromosomal gene replacement mutagenesis, up to all five of the following peptidase genes were inactivated (fivefold mutant): pepX, pepO, pepT, pepC, and pepN. Multiple mutations led to slower growth rates in milk, the general trend being that growth rates decreased when more peptidases were inactivated. The fivefold mutant grew more than 10 times more slowly in milk than the wild-type strain. In one of the fourfold mutants and in the fivefold mutant, the intracellular pools of amino acids were lower than those of the wild type, whereas peptides had accumulated inside the cell. No significant differences in the activities of the cell envelope-associated proteinase and of the oligopeptide transport system were observed. Also, the expression of the peptidases still present in the various mutants was not detectably affected. Thus, the lower growth rates can directly be attributed to the inability of the mutants to degrade casein-derived peptides. These results supply the first direct evidence for the functioning of lactococcal peptidases in the degradation of milk proteins. Furthermore, the study provides critical information about the relative importance of the peptidases for growth in milk, the order of events in the proteolytic pathway, and the regulation of its individual components.     

Influence of the yeast strain on the changes of the amino acids,peptides and proteins during sparkling wine production by the traditional method

The influence of five yeast strains on the nitrogen fractions, amino acids, peptides and proteins, during 12 months of aging of sparkling wines produced by the traditional or Champenoise method, was studied. High-performance liquid chromatography (HPLC) techniques were used for analysis of the amino acid and peptide fractions. Proteins plus polypeptides were determined by the colorimetric Bradford method. Four main stages were detected in the aging of wines with yeast. In the first stage, a second fermentation took place; amino acids and proteins plus polypeptides diminished, and peptides were liberated. In the second stage, there was a release of amino acids and proteins, and peptides were degraded. In the third stage, the release of proteins and peptides predominated. In the fourth stage, the amino acid concentration diminished. The yeast strain used influenced the content of free amino acids and peptides and the aging time in all the nitrogen fractions. Received 25 March 2002/ Accepted in revised form 31 July 2002     

Pathway for degradation of peptides generated by proteasomes: a key role for thimet oligopeptidase and other metallopeptidases

The degradation of cellular proteins by proteasomes generates peptides 2-24 residues long, which are hydrolyzed rapidly to amino acids. To define the final steps in this pathway and the responsible peptidases, we fractionated by size the peptides generated by proteasomes from beta-[14C]casein and studied in HeLa cell extracts the degradation of the 9-17 residue fraction and also of synthetic deca- and dodecapeptide libraries, because peptides of this size serve as precursors to MHC class I antigenic peptides. Their hydrolysis was followed by measuring the generation of smaller peptides or of new amino groups using fluorescamine. The 14C-labeled peptides released by 20 S proteasomes could not be degraded further by proteasomes. However, their degradation in the extracts and that of the peptide libraries was completely blocked by o-phenanthroline and thus required metallopeptidases. One such endopeptidase, thimet oligopeptidase (TOP), which was recently shown to degrade many antigenic precursors in the cytosol, was found to play a major role in degrading proteasome products. Inhibition or immunodepletion of TOP decreased their degradation and that of the peptide libraries by 30-50%. Pure TOP failed to degrade proteasome products 18-24 residues long but degraded the 9-17 residue fraction to peptides of 6-9 residues. When aminopeptidases in the cell extract were inhibited with bestatin, the 9-17 residue proteasome products were also converted to peptides of 6-9 residues, instead of smaller products. Accordingly, the cytosolic aminopeptidase, leucine aminopeptidase, could not degrade the 9-17 residue fraction but hydrolyzed the peptides generated by TOP to smaller products, recapitulating the process in cell extracts. Inactivation of both TOP and aminopeptidases blocked the degradation of proteasome products and peptide libraries nearly completely. Thus, degradation of most 9-17 residue proteasome products is initiated by endoproteolytic cleavages, primarily by TOP, and the resulting 6-9 residue fragments are further digested to amino acids by aminopeptidases.     

Degradation of HSA-AX15(R13K) when expressed in Pichia pastoris can be reduced via the disruption of YPS1 gene in this yeast

Expression of recombinant protein HSA-AX15(R13K) in Pichia pastoris GS115 strain produced both the intact protein and its two degradation products with molecular weights of around 43kDa and 66.2kDa, respectively. To reduce or avoid the degradation, a modified P. pastoris GS115 stain, in which YPS1 gene was disrupted, was constructed via homologous recombination and used as a host strain for the HSA-AX15(R13K) expression. After 60h of induction during culture, it was found that the degradation product of around 66.2kDa was reduced significantly in the supernatant of yps1-disrupted strain compared with that in the supernatant of wild-type strain. By the Western blot analysis of culture supernatants from wild-type and yps1-disrupted strains expressing HSA-AX15(R13K), the significant improvement was also seen in the degradation product of around 43kDa. Comparison of cell growth between the two strains demonstrated a similar growth tendency, thereby indicating that the disruption of YPS1 gene has no effect on the normal physiology of GS115 strain. Following induction for 60h, the yield of intact HSA-AX15(R13K) in the yps1 disruptant was three-fold higher than that in the wild-type strain. Therefore, such a P. pastoris mutant deficient in YPS1 activity is suitable for the high-level expression of recombinant protein HSA-AX15(R13K).     

Deoxyribonucleic Acid Repair in a Highly Radiation-Resistant Strain of Salmonella typhimurium

Deoxyribonucleic acid repair was studied in gamma-irradiated wild-type Salmonella typhimurium and in a radiation-resistant derivative 20 times more resistant than wild type. After exposure to 20 or 50 krad, the wild-type strain (DB21) degraded 30 to 50% of its prelabeled DNA into acid-soluble fragments, whereas the radioresistant strain degraded less than 15% after 4 h of incubation. Post-irradiation synthesis of DNA in the wild-type strain DB21 was reduced after a dose of 20 krad and totally inhibited after exposure to 200 krad. With radiation-resistant strain, D21R6008, on the other hand, DNA synthesis was delayed after a dose of 200 krad but not inhibited. Doses of 20 and 200 krad produced a similar number of single-strand breaks in the DNA of both strains as determined by zone sedimentation analysis in alkaline sucrose gradients. The radiation-resistant strain D21R6008, on the other hand, DNA synthesis was strand breaks in its DNA and repairs these damages more rapidly than wild-type Salmonella.     

Abnormal proteins of shortened length are preferentially degraded in the cytosol of cultured MRC5 fibroblasts

Puromycyl peptides were degraded in MRC5 fibroblasts more rapidly than normal proteins labelled for the corresponding length of time for both long and short labelling periods. The degradation of the puromycyl peptides occurred almost exclusively in the cytosol of the cells. Even when the half-lives of normal and puromycyl peptides were manipulated to be similar, proportionally more of the normal proteins were degraded in the lysosomes. The rapid degradation of the puromycyl peptides was not due to the inhibition of protein synthesis brought about by puromycin but was due to the structure of the substrates themselves. The degree and intracellular site of degradation of puromycyl peptides closely mimic those of abnormal (missense) proteins containing amino acid analogues.     

Isolation and characterization of a bacteriophage T5 mutant deficient in deoxynucleoside 5''-monophosphatase activity.

A bacteriophage T5 mutant has been isolated that is completely deficient in the induction of deoxynucleoside 5'-monophosphatase activity during infection of Escherichia coli F. The mutant bacteriophage has been shown to be deficient in the excretion of the final products of DNA degradation during infection of E. coli F, and about 30% of the host DNA's thymine residues were reinocorporated into phage DNA. During infection with this mutant, host DNA degradation to trichloroacetic acid-soluble products was normal, host DNA synthesis was shut off normally, and second-step transfer was not delayed. However, induction of early phage enzymes and production of DNA and phage were delayed by 5 to 15 min but eventually reached normal levels. The mutant's phenotype strongly suggests that the enzyme's role is to act at the final stage in the T5-induced system of host DNA degradation by hydrolyzing deoxynucleoside 5'-monophosphates to deoxynucleosides and free phosphate; failure to do this may delay expression of the second-step-transfer DNA.     

Oxygen-insensitive nitroreductases of Escherichia coli do not reduce 3-nitrotyrosine

The oxygen-insensitive nitroreductases nfsA and nfsB are known to reduce para-nitrated aromatic compounds. We tested the hypothesis that these nitroreductases are capable of reducing 3-nitrotyrosine in proteins and peptides, as well as in free amino acids using wild-type and nfsA nfsB mutant strains of Escherichia coli. E. coli homogenates were incubated with nitrated proteins and the level of 3-nitrotyrosine immunoreactivity was assayed by Western blotting. Assay conditions that allow the nitroreductases to rapidly reduce nitrofurantoin did not result in the modification of 3-nitrotyrosine in protein, peptide, or free amino acid. Stimulation of nfsA nfsB activity with paraquat had no effect on 3-nitrotyrosine reduction. Nonlethal exposure of E. coli to peroxynitrite/CO(2) resulted in the reproducible nitration of tyrosine residues in endogenous proteins. The degree of 3-nitrotyrosine immunoreactivity over the 2-h postexposure period did not differ between mutant and wild-type strains. These results indicate that the nfsA and nfsB enzymes do not reduce 3-nitrotyrosine.     

Oligopeptides are the main source of nitrogen for Lactococcus lactis during growth in milk.

The consumption of amino acids and peptides was monitored during growth in milk of proteinase-positive (Prt+) and -negative (Prt-) strains of Lactococcus lactis. The Prt- strains showed monophasic exponential growth, while the Prt+ strains grew in two phases. The first growth phases of the Prt+ and Prt- strains were in same, and no hydrolysis of casein was observed. Also, the levels of consumption of amino acids and peptides in the Prt+ and Prt- strains were similar. At the end of this growth phase, not all free amino acids and peptides were used, indicating that the remaining free amino acids and peptides were unable to sustain growth. The consumption of free amino acids was very low (about 5 mg/liter), suggesting that these nitrogen sources play only a minor role in growth. Oligopeptide transport-deficient strains (Opp-) of L. lactis were unable to utilize oligopeptides and grew poorly in milk. However, a di- and tripeptide transport-deficient strain (DtpT-) grew exactly like the wild type (Opp+ Dtpt+) did. These observations indicate that oligopeptides represent the main nitrogen source for growth in milk during the first growth phase. In the second phase of growth of Prt+ strains, milk proteins are hydrolyzed to peptides by the proteinase. Several of the oligopeptides formed are taken up and hydrolyzed internally by peptidases to amino acids, several of which are subsequently released into the medium (see also E.R.S. Kunji, A. Hagting, C.J. De Vries, V. Juillard, A.J. Haandrikman, B. Poolman, and W.N. Konings, J. Biol. Chem. 270:1569-1574, 1995).(ABSTRACT TRUNCATED AT 250 WORDS)     

Stabilization of glucose-starved Escherichia coli K12 and Salmonella typhimurium LT2 by peptidase-deficient mutants

Escherichia coli K12 and Salmonella typhimurium LT2 cells were stabilized during carbon starvation in the presence of peptidase-deficient mutant strains. The rate of loss of viability of the wild-type S. typhimurium strain was decreased an average of 2-fold, and the rate for the wild-type E. coli strain was decreased about 2.3-fold, when either was starved in the presence of the multiply peptidase-deficient S. typhimurium strain TN852; other peptidase-deficient strains exhibited similar stabilizing effects. Starving wild-type S. typhimurium LT2 cells utilized peptides excreted by the starving peptidase-deficient cells for protein synthesis, and, to a lesser extent, as respiratory substrates. Provision of free amino acids in steady-state levels to starving E. coli K12 cells in a cell recycle apparatus had a stabilizing effect similar to that of mixing with peptidase-deficient cells.     

Degradation and regurgitation of extracellular proteins by cultured mouse peritoneal macrophages and baby hamster kidney fibroblasts. Kinetic evidence that the transfer of proteins to lysosomes is not irreversible

The uptake and degradation of bovine serum albumin (BSA), bovine liver catalase, and rabbit muscle enolase have been studied in cultured mouse peritoneal macrophages (MPM) and baby hamster kidney fibroblasts (BHK cells). Rates constant for the uptake of the three proteins by MPM were similar. In addition, BSA accumulation was independent of BSA concentration in the uptake medium and was not inhibited by a large excess of serum, suggesting that protein accumulation was by fluid phase pinocytosis. Following an overnight uptake, 20-30% of the accumulated protein was subsequently regurgitated into the medium in a trichloroacetic acid/phosphotungstic acid-precipitable form. This material co-migrated with the authentic protein during molecular sieve chromatography on Sephadex G-50. The rates of appearance of trichloroacetic acid/phosphotungstic acid-insoluble products were greater than expected for cell death and leakage. The observed first order rate constants, kobs, for the appearance of trichloroacetic acid/phosphotungstic acid-soluble and trichloroacetic acid/phosphotungstic acid-insoluble products in the culture medium were identical, indicating that both products were released in parallel from MPM and BHK cells. The kobs for intracellular BSA degradation and regurgitation were independent of the initial BSA concentration in the uptake medium, but were decreased about 35% when degradation was allowed to proceed in the presence of high concentrations of serum. Degradation was also inhibited by chloroquine and pepstatin. Inhibition of degradation was accompanied by an increase in the total amount of regurgitated protein appearing in the medium. Remarkably, however, these inhibitors also decreased kobs for regurgitation, thereby preserving the similarity in the observed rate constants for the appearance of trichloroacetic acid/phosphotungstic acid-soluble and trichloroacetic acid/phosphotungstic acid-insoluble products. These and other results were inconsistent with desorption of proteins from the surface of the culture dish or the surface of cells as the source of the trichloroacetic acid/phosphotungstic acid-insoluble label appearing in the medium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of the mammalian mitochondrial genome. Role for membrane potential in the production of mature translation products

Protein synthesis was investigated in isolated mitochondria under conditions which either inhibited electron transport or uncoupled oxidative phosphorylation. In a medium containing an exogenous source of ATP and oligomycin, an inhibitor of the ATP synthase complex, incorporation of [35S]methionine into proteins is stimulated in the presence of inhibitors of the electron transport chain; substituting uncouplers of oxidative phosphorylation for the latter leads, in contrast, to a decrease in the rate of incorporation of the labeled amino acid into mitochondrial translation products. Studies on the metabolic stability of mitochondrial translation products revealed that "mature" polypeptides made in isolated mitochondria are stable as indicated by the absence of degradation during a 50 min "chase" period. Under conditions which reduce or dissipate the membrane potential, 50-60% of the newly made polypeptides (pulse) are degraded within 50 min. The kinetics of the degradation process for individual mitochondrial gene products reveal that the largest proportion of polypeptides degraded to an acid-soluble form during the chase period are abnormal proteins, likely the result of premature chain termination. Emerging as a common denominator in these studies is a role for a transmembrane potential across the inner membrane in the production of mature "stable" mitochondrial gene products.     

Divergent effects of a dnaK mutation on abnormal protein degradation in Escherichia coli

Escherichia coli bacteria produce at least one 70 kD stress protein, the product of the dnaK gene. We have compared the rates of degradation of different types of abnormal proteins in null Ion E. coli with a partial deletion of the dnaK gene with the rates observed in null Ion dnaK+ cells. We have found that both canavanyl proteins and puromycyl polypeptides are degraded more slowly in the null dnaK mutants than in the dnaK+ strain. However, a temperature-sensitive mutant LacI protein is degraded more rapidly in the null dnaK strain. The stability of this temperature-sensitive LacI protein was also examined in detail under various other conditions.