Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
Correspondence
Dipankar Nandi
nandi{at}biochem.iisc.ernet.in
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ABSTRACT |
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INTRODUCTION |
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Recently, characterizing enzymes involved in the ATP-independent process and studying their functional role has gained importance. Many of the studies have been done on archaeal and eukaryotic organisms. Enzymes involved in downstream processing, for example, tripeptidyl peptidase II, thimet oligopeptidase, bleomycin hydrolase, leucine aminopeptidase and puromycin-sensitive aminopeptidase, are also important in the trimming and degradation of major histocompatibility complex (MHC) class I binding peptides in mammals (Beninga et al., 1998; Stoltze et al., 2000
; Saric et al., 2001
; York et al., 2003
). In T. acidophilum, this proteolytic process has been reconstituted in vitro (Tamura, N. et al., 1998
) and crystal structures have been determined for most of the key enzymes (Lowe et al., 1995
; Brandstetter et al., 2001
; Goettig et al., 2002
). Peptides released by 20S proteasomes are further degraded by Tricorn endoprotease to short peptides which are in turn broken down into amino acids by the Tricorn interacting exopeptidases, F1, F2 and F3. In fact, a model of the TricornF1 complex suggests that the
7 propeller of Tricorn is used for substrate entry whereas the
6 propeller is for product egress and may act as a docking site for binding the Tricorn interacting factor F1 (Goettig et al., 2002
). Methionine aminopeptidases are essential in E. coli and Saccharomyces cerevisiae, demonstrating that cleavage of the N-terminal methionine from some proteins is critical for cellular function (Chang et al., 1989
; Bradshaw et al., 1998
). Peptidases in Lactococcus lactis are required for proteolysis and growth in milk (Mierau et al., 1996
). The turnover of cellular proteins is reduced in multiple-peptidase mutants, but not in single-peptidase mutants, in Salmonella typhimurium (Yen et al., 1980
) and E. coli (Miller & Schwartz, 1978
; Conlin & Miller, 1995
). Due to the redundancy of peptidases in prokaryotes, there is little information available on the physiological roles of these enzymes.
PepN, also known to be the sole alanine aminopeptidase in E. coli, was identified 28 years ago (Lazdunski et al., 1975a, b
). Recently, we initiated studies on enzymes involved in downstream processing in eubacteria. In the course of our studies, we identified PepN from E. coli to be responsible for cleaving Suc-LLVY-7-amido-4-methylcoumarin (AMC), a substrate cleaved by the 20S proteasome from all sources. PepN was further characterized with respect to its aminoendopeptidase activities (Chandu et al., 2003
). PepN and its homologues are well conserved in all kingdoms and play a role in downstream processing during cytosolic protein degradation. We wished to study, using synthetic peptidase substrates, the substrate specificity of pure PepN. In addition, we wished to evaluate the contribution of PepN to overall cellular peptidase activities in extracts of wild-type and pepN mutant strains of E. coli. Previous studies had failed to demonstrate a phenotype for E. coli strains lacking pepN (McCaman et al., 1982
; Bally et al., 1983
). Therefore, we wished to investigate the role of PepN during different stress conditions. In this study, we demonstrate that PepN is responsible for the majority of the aminopeptidase activity in E. coli and that it prefers to cleave basic and small amino acids at the amino terminus of substrates. Furthermore, we show that PepN plays a negative role during sodium salicylate (NaSal)-induced stress.
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METHODS |
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Generation of antiserum to PepN and Western analysis.
Antiserum was raised against purified PepN by injecting 200 µg of the pure protein per rabbit as an emulsion with complete Freund's adjuvant subcutaneously. Two booster injections of 100 µg PepN per rabbit were given with incomplete Freund's adjuvant, after gaps of 2 weeks. One week after the second booster, the serum was obtained and stored at -70 °C until further use. This antiserum against PepN specifically detects the enzyme from purified preparations as well as crude extracts, by both ELISA and Western blotting. However, pre-immune serum collected from the same rabbit before PepN immunization displayed negligible reactivity (data not shown). Western analysis was performed by separating the cellular extracts on a 10 % SDS-PAGE gel and transferring them to a nitrocellulose membrane. After blocking overnight with 1 % gelatin in 50 mM PBS (0·01 M NaH2PO4, 0·04 M Na2HPO4, 0·15 M NaCl, pH 7·4), the pre-immune serum and PepN-specific antiserum were used at a dilution of 1 : 10 000. Goat anti-rabbit antibody conjugated to horseradish peroxidase (Bangalore Genei) was used as secondary antibody (1 : 2000 dilution). Antibodies bound to PepN were visualized by staining with hydrogen peroxide and 3,3'-diaminobenzidine (Sigma) in 50 mM PBS. This antiserum specifically recognized PepN in cytosolic extracts from wild-type E. coli but not in cytosolic extracts from two strains lacking pepN, namely, 9218 and DH5pepN (Fig. 2a, b
). Also, no cross-reactivity was found in cytosolic extracts from M. smegmatis or mouse liver with this antiserum (data not shown).
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NaSal-induced stress.
Single colonies of different E. coli strains were grown in 3 ml LB medium overnight, with appropriate antibiotics. Cell-free extracts of the strains were prepared after sonication and assayed for hydrolysis of Suc-LLVY-AMC or L-Ala-pNA, to confirm the authenticity of the cultures, as a standard practice. Each tested culture was streaked on one quadrant of appropriate plates and incubated at 37 °C in the absence or presence of increasing amounts of NaSal. In addition, a plate streaked with different strains was incubated at 42 °C to study the effect of growth at high temperature. After growth for 10 h, the images of plates were obtained with the ALPHADIGIDOC documentation system (San Leandro, CA, USA).
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RESULTS |
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Endopeptidase substrate hydrolysis profile of pepN-deficient E. coli extracts
Next, we studied the contribution of PepN to the overall endopeptidase activity in E. coli. The hydrolysis of a panel of endopeptidase substrates was studied in cytosolic extracts of the two pepN mutants, 9218 and DH5pepN, and their respective wild-type strains, K-12 RV and DH5
(Fig. 3
a). The hydrolysis of three endopeptidase substrates, Suc-LLVY-AMC, Suc-AAF-AMC and Boc-LRR-AMC (partial effect), was reduced (Fig. 3a
) in extracts of both pepN mutants. Overexpression of PepN in E. coli 9218 with pBM15 rescued all three activities (Fig. 3b
). The rescue of Boc-LRR-AMC was partial as PepN and another uncharacterized serine peptidase (data not shown) were responsible for its hydrolysis. Although we were unable to infer any cleavage preference for endopeptidase substrates by PepN, these results demonstrated that PepN is responsible for hydrolysis of some endopeptidase substrates. As both the mutants were generated in different genetic backgrounds, effects that were observed in both mutants only were considered as significant.
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DISCUSSION |
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Previously, PepN was known to cleave Ala, Lys, Gly, Leu (McCaman & Villarejo, 1982) and the dipeptide cysteinylglycine (Suzuki et al., 2001
). The wide range of substrates used in this study has not been used before and our systematic analysis of the cleavage preference of PepN revealed that it is a broad-specificity aminopeptidase, which cleaves basic and small amino acids substrates better than others (Fig. 1
). There are few enzymes that can cleave proline-containing peptides; this activity is performed by dedicated enzymes, known as proline iminopeptidases and prolidases (Vanhoof et al., 1995
). Interestingly, PepN cleaves L-Pro-
NA, a proline aminopeptidase substrate. Although PepN is known as the sole alanine aminopeptidase, it cleaves arginine better than alanine. However, the alanine/arginine aminopeptidase-1 (Aap-1), the PepN homologue in Saccharomyces cerevisiae, cleaves both Arg and Ala (Caprioglio et al., 1993
). PepN homologues from L. lactis (van Allen-Boerrigter et al., 1991
), Streptococcus thermophilus (Chavagnat et al., 1999
) and Aspergillus niger (Basten et al., 2001
) are, primarily, lysine aminopeptidases. In general, aminopeptidases involved in protein degradation act on short, not long, peptides (Tamura, N. et al., 1998
; Franzetti et al., 2002
). It is possible that PepN preferentially cleaves peptides with basic or small amino acids as amino-terminal residues. Given the specificity of PepN, it is interesting to speculate that, perhaps, other aminopeptidases may be specific for hydrolysis of acidic amino acids, for example, PepB and/or PepE (Larsen et al., 2001
). This hypothesis may be justified as the cleavage specificities of aminopeptidases F1, F2 and F3 in T. acidophilum are distinct: F1 is a proline iminopeptidase, F2 prefers basic amino acid substrates and F3 prefers acidic amino acid substrates (Tamura, N. et al., 1998
).
Although there is some information on the role of ATP-dependent proteases, for example, Lon and Clp, under stress conditions (Gottesman, 1996; Kuroda et al., 2001
), not much is known about the physiological role of ATP-independent peptidases. PepA, PepB, PepD and PepN are thought to be redundant and deficiency in all four peptidases is required to demonstrate an effect on cytosolic protein turnover (Miller & Schwartz, 1978
; Yen et al., 1980
). There is a linear relationship between PepN activity and bacterial growth (Bally et al., 1983
) and pepN transcription is induced on phosphate starvation, anaerobic conditions and growth in minimal medium (Gharbi et al., 1985
). As the significance of these observations is unclear, we resorted to a genetic approach to study the role of PepN under different conditions. The observation that the pepN transcript is induced on NaSal treatment (Pomposiello et al., 2001
), but not heat shock (Richmond et al., 1999
), prompted us to study the role of PepN under different stress conditions. As shown in Fig. 6
, the lack of PepN resulted in increased growth, whereas PepN expression clearly reduced the growth of E. coli, in the presence of NaSal. Thus, PepN is a negative regulator of NaSal-induced stress. The observation that pepN is induced on NaSal treatment and our current result that PepN is a negative regulator may at first appear to be inconsistent. However, modulation of gene expression, especially microarray data, needs to be confirmed using genetic and/or biochemical approaches (Slonim, 2002
). Also, NaSal inactivates the repressor marR and activates marA; however, both these genes are induced on NaSal treatment (Pomposiello et al., 2001
). The use of genetic mutants and overexpression studies, as shown in Fig. 6
, is the appropriate approach to address the role of pepN under stress. NaSal does not modulate the activity of pure PepN and no difference in PepN activity was found in extracts of untreated or NaSal-treated E. coli (data not shown). Notably, pepN homologues in other organisms are known to play distinct roles. L. lactis lacking pepN displayed growth reduction by 20 % in medium containing casein as carbon source (Mierau et al., 1996
). Saccharomyces cerevisiae lacking aap-1, a pepN homologue, accumulates less glycogen, whereas, on transforming
aap-1 cells with aap-1, there is more glycogen accumulation (Caprioglio et al., 1993
). As glycogen accumulation occurs just as glucose is being exhausted during diauxic growth, glycogen accumulation is considered a marker for stress in yeast. Male mice lacking puromycin-sensitive aminopeptidase, a mammalian PepN homologue, are sterile due to impaired spermatogenesis and degenerative morphology of Sertoli cells (Osada et al., 2001a
). Female mice lacking puromycin-sensitive aminopeptidase are also sterile due to impaired formation of the corpus luteum (Osada et al., 2001b
). PepN and its homologues play distinct roles in the physiology of organisms from different kingdoms. Thus, enzymes involved in downstream processing may play specialized roles in cellular processes.
In mammals, NaSal acts as a non-steroidal anti-inflammatory agent. It is an inducer of heat shock and inhibits the production of inflammatory cytokines, whereas in plants it is part of the host defence system (Price et al., 2000). In prokaryotes, NaSal is used to study the response to a xenobiotic compound. There are several mechanisms by which NaSal acts dissipation of the proton gradient across the inner membrane, iron chelation, growth inhibition, induction of heat shock and the marA regulon resulting in the modulation of several genes (Price et al., 2000
; Pomposiello et al., 2001
). NaSal binds and inactivates MarR and activates MarA, resulting in changes in the outer-membrane profile, decreased permeability to antibiotics and, consequently, increased antibiotic resistance (Cohen et al., 1993
; Ramani & Boakye, 2001
). To understand the cellular role of PepN and its relation to certain stress conditions (induced by NaSal, etc.), it is important to consider that it acts as an aminoendopeptidase. In terms of protein turnover, the aminopeptidase function of PepN will be clearly important in the recycling of amino acids. However, we know that there are redundant aminopeptidases which can take over this role (Miller & Schwartz, 1978
; Yen et al., 1980
). Therefore, during stress the aminopeptidase activity may be important in cleaving peptides or proteins containing basic or small amino acids which reduce the ability of E. coli to display resistance to stress. Arginine and lysine at the amino terminus destabilize proteins and such proteins are targeted for degradation (Gonzales & Robert-Baudouy, 1996
). Also, aminopeptidase action may result in isoforms of proteins possessing different amino terminal amino acids (Ishino et al., 1987
). Alternatively, PepN via its endopeptidase activity may modulate the proteome of cells. In fact, PepN is required for the activation of the antibiotic albomycin inside E. coli; therefore, pepN mutant cells are resistant to albomycin action. The predicted cleavage sites in albomycin suggest that the endopeptidase activity of PepN plays a role in this cleavage (Braun et al., 1983
). It is possible that PepN cleaves cellular proteins that result in decreased ability of E. coli to resist NaSal-mediated stress. However, in the absence of PepN, these proteins may be present and help to withstand NaSal-induced stress. Comparison of the proteome of wild-type and pepN mutant strains may identify such cellular substrates of PepN. Further studies are in progress to understand the mechanisms by which the major aminopeptidase in E. coli, PepN, functions as an aminoendopeptidase to negatively modulate the response to NaSal-induced stress.
Thus far, seven aminoendopeptidases have been reported: -N-benzoylarginine-
-naphthylamide hydrolase (Singh & Kalnitsky, 1980
), hydrolase H (Okitani et al., 1981
; Nishimura et al., 1983
), PepN (Chandu et al., 2003
), bleomycin hydrolase (Koldamova et al., 1998
), cathepsin H (Turk et al., 2001
), multicorn (Osmulski & Gaczynska, 1998
) and tripeptidyl peptidase II (Geier et al., 1999
). Of these, the last five are involved in cytosolic protein degradation, suggesting an important role for these enzymes in this process. Notably, PepN is the only bacterial aminoendopeptidase characterized (Chandu et al., 2003
). PepN is a better aminopeptidase than an endopeptidase, as indeed are other aminoendopeptidases, for example, multicorn (Osmulski & Gaczynska, 1998
) and tripeptidyl peptidase II (Geier et al., 1999
). Although purified enzymes involved in downstream processing have been studied in T. acidophilum (Tamura, N. et al., 1998
), Schizosaccharomyces pombe (Osmulski & Gaczynska, 1998
) and mouse cells (Geier et al., 1999
), there are no studies reported in organisms lacking these enzymes. The extensive substrate preference profile of pure PepN and its role in cellular extracts, together with its physiological role during some stress conditions, are unique to this study and will enhance our understanding of the role of the aminoendopeptidase PepN in cytosolic protein degradation in E. coli.
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ACKNOWLEDGEMENTS |
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Received 27 May 2003;
revised 29 July 2003;
accepted 13 August 2003.
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