Functional characterization of PepN, an aminoendopeptidase in escherichia coli, and its implications in cytosolic protein degradation

Abstract

Functional Characterization of PepN, an Aminoendopeptidase in Escherichia coli, and Its Implications in Cytosolic Protein Degradation

Steady-state levels of cellular proteins are maintained by a balance between their rates of synthesis and degradation. While protein synthesis has been extensively studied, the mechanisms and molecules involved in protein degradation have only recently begun to be fully appreciated. Increasing evidence indicates that intracellular protein degradation plays important roles in regulating major metabolic pathways, the cell cycle, transcription, antigen processing, oncogenesis, Alzheimer’s disease, cystic fibrosis, and other processes.

The protein degradation pathway can be divided into two distinct steps: (i) ATP-dependent unfolding and targeting of polypeptides destined for hydrolysis into oligopeptides, and (ii) ATP-independent downstream processing of these oligopeptides into amino acids.

In eukaryotes, 26S proteasomes (~2000 kDa), consisting of catalytic 20S proteasomes (~650 kDa) bound to 19S regulatory complexes, are responsible for the ATP-dependent step of cytosolic protein degradation. True 20S proteasomes are absent in most eubacteria (e.g., E. coli). However, complete genome sequences of various eubacteria have revealed the presence of proteasomal genes in a subgroup, the Actinomycetales, to which Mycobacterium belongs.

To initiate studies on protein degradation in Mycobacterium smegmatis, cellular proteins were fractionated by sucrose density gradient ultracentrifugation, and fractions were tested for peptidase activity using synthetic fluorogenic peptide substrates. Of the four substrates tested, Suc-LLVY-AMC is cleaved by 20S proteasomes from all known sources. Two peaks of Suc-LLVY-AMC hydrolyzing activity were observed: one at ~100 kDa and another at ~650 kDa. Preliminary studies suggested that the ~100 kDa low-molecular-weight (LMW) peptidase, which shares substrate specificity with 20S proteasomes, is a zinc metallopeptidase. Surprisingly, LMW peptidase activity was also detected in E. coli. Using the annotated E. coli genome sequence and available literature on E. coli peptidases, two probable candidates (pepN and pqqL) were identified.

Genetic studies using an E. coli mutant (strain 9218) and mass spectrometry-based sequencing of the biochemically purified LMW enzyme from M. smegmatis confirmed that PepN is responsible for Suc-LLVY-AMC hydrolyzing activity. E. coli PepN was purified ~35-fold using a combination of ion-exchange and hydrophobic interaction chromatography steps, and optimal assay conditions were standardized. Genetic and biochemical studies demonstrated that PepN is an aminoendopeptidase. A single band was observed after silver staining of SDS-PAGE gels, and aminopeptidase and endopeptidase activities displayed similar profiles following gel filtration, thermal denaturation, and inhibition by a panel of protease inhibitors. However, differences in sensitivity of these two activities to inhibition by bestatin, an aminopeptidase-specific inhibitor, were observed. Finally, induced expression of cloned pepN in DH5pepN, a targeted pepN-deletion mutant, rescued both aminopeptidase and endopeptidase activities.

An antiserum raised against purified PepN displayed a high titer but did not inhibit either aminopeptidase or endopeptidase activity of purified PepN. Western blot analysis revealed that strain 9218, an N-methyl-N-nitro-N-nitrosoguanidine (NTG)-derived mutant used to identify PepN, did not express PepN. Overexpression of pepN from strain 9218 was unable to compensate for the loss of PepN activity in DH5pepN. Sequence analysis of pepN from strain 9218 revealed a transversion of 228G to 228T, resulting in replacement of Glu77 with a stop codon.

To understand the contribution of PepN to peptidase activity in E. coli extracts, hydrolysis of a panel of endopeptidase and aminopeptidase substrates was studied in extracts from wild-type strains and two PepN mutants, 9218 and DH5pepN. Hydrolysis of three out of eight endopeptidase substrates tested was reduced in pepN mutant extracts and was restored by PepN overexpression. Similarly, hydrolysis of ten out of fourteen exopeptidase substrates was greatly reduced in both pepN mutants. Transformation with a PepN overexpression construct complemented the hydrolysis of the affected aminopeptidase substrates. These results suggest that PepN is responsible for the majority of aminopeptidase activity in E. coli.

Further studies with purified PepN revealed a preference for cleaving basic and small amino acids as aminopeptidase substrates. Kinetic characterization demonstrated that PepN cleaves arginine approximately eightfold more efficiently than alanine, although it had previously been characterized as the sole alanine aminopeptidase in E. coli. Since most aminopeptidases cannot cleave long peptides or intact proteins, PepN was tested for its ability to hydrolyze such substrates using its aminoendopeptidase activity. PepN hydrolyzed oxidized insulin B-chain peptide in a time-dependent manner and cleaved casein, a known protease substrate, in a bestatin-independent fashion. Kinetic analysis showed that PepN cleaves substrates in the following order of preference:

aminopeptidase substrates > endopeptidase substrates > protease substrates.

To investigate the role of PepN in cellular metabolism, the ability of E. coli DH5 and DH5pepN, with or without PepN overexpression, to grow under selected stress conditions was examined. Although no significant differences were observed under most stress conditions tested, PepN appeared to act as a negative regulator under sodium salicylate-induced stress.

Complete genome sequences from diverse organisms provide opportunities to explore the roles of newly identified enzymes and their homologues. In this context, key enzymes involved in various steps of cytosolic protein degradation in E. coli were selected for analysis. Genome analysis was performed to identify homologues of these enzymes, followed by multiple sequence alignment of active-site residues from seventeen organisms belonging to thirteen genera, including representatives from Mycoplasma, eubacteria, archaebacteria, and eukaryotes.

The presence of Lon, PepA, PepB, PepM, PepP, and PepQ homologues in the Mycoplasma genome reflects the importance of proteases and peptidases as part of the minimal gene set required for survival. Some homologues of E. coli peptidases perform specialized functions in eukaryotes, such as processing MHC class I-binding peptides.

PepN and its homologues are evolutionarily conserved. This conservation, together with the functional characteristics elucidated in this study, suggests that they play an important role in intracellular protein degradation. Sequence-based homologues of PepN include Tricorn-interacting factors F2 and F3 in archaea and puromycin-sensitive aminopeptidase in mammals. However, biochemical characteristics suggest that PepN, through its aminoendopeptidase activity, may be functionally similar to enzymes involved in downstream processing of proteins in the cytosol, such as the Tricorn/F1/F2/F3 complex in archaea and tripeptidyl peptidase II (TPPII) in eukaryotes.

Putative sequence-based homologues of known downstream-processing enzymes from representative organisms across different kingdoms were identified, and alignment of active-site residues was performed. Homologues of endopeptidases and some oligopeptidases were not observed in many representative organisms, suggesting specificity during the initial steps of downstream processing. However, aminopeptidase homologues were readily found in most organisms. These findings suggest that specific endoproteases and peptidases are required during the proximal steps of protein degradation in different organisms, whereas peptidases involved in later stages of downstream processing are more conserved across kingdoms.

In summary, this study, through biochemical and genetic evidence, identified PepN—previously characterized as an alanine aminopeptidase—as responsible for the majority of aminopeptidase activity in E. coli. In addition, PepN acts as a negative regulator under sodium salicylate-induced stress. PepN is an aminoendopeptidase and may function as the eubacterial homologue of downstream-processing enzymes in archaea and eukarya.