Characterization of Two New Aminopeptidases in
            <i>Escherichia coli</i>

Zheng, Yu; Roberts, Richard J.; Kasif, Simon; Guan, Chaowen

doi:10.1128/jb.187.11.3671-3677.2005

Cited by 14 publications

(17 citation statements)

References 25 publications

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“…Yet according to our results on the distribution of M42 aminopeptidases, E. coli also possesses three M42 aminopeptidases. The activity of one of them, YpdE, has been characterized previously [51], and it is found in a dodecameric state (unpublished data). Why this species maintains several complexes capable of degrading peptides has not yet been studied.…”

Section: Discussionmentioning

confidence: 99%

Functional Characterization of Two M42 Aminopeptidases Erroneously Annotated as Cellulases

Dutoit¹,

Brandt²,

Legrain³

et al. 2012

PLoS ONE

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Several aminopeptidases of the M42 family have been described as tetrahedral-shaped dodecameric (TET) aminopeptidases. A current hypothesis suggests that these enzymes are involved, along with the tricorn peptidase, in degrading peptides produced by the proteasome. Yet the M42 family remains ill defined, as some members have been annotated as cellulases because of their homology with CelM, formerly described as an endoglucanase of Clostridium thermocellum. Here we describe the catalytic functions and substrate profiles CelM and of TmPep1050, the latter having been annotated as an endoglucanase of Thermotoga maritima. Both enzymes were shown to catalyze hydrolysis of nonpolar aliphatic L-amino acid-pNA substrates, the L-leucine derivative appearing as the best substrate. No significant endoglucanase activity was measured, either for TmPep1050 or CelM. Addition of cobalt ions enhanced the activity of both enzymes significantly, while both the chelating agent EDTA and bestatin, a specific inhibitor of metalloaminopeptidases, proved inhibitory. Our results strongly suggest that one should avoid annotating members of the M42 aminopeptidase family as cellulases. In an updated assessment of the distribution of M42 aminopeptidases, we found TET aminopeptidases to be distributed widely amongst archaea and bacteria. We additionally observed that several phyla lack both TET and tricorn. This suggests that other complexes may act downstream from the proteasome.

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Section: Discussionmentioning

confidence: 99%

Functional Characterization of Two M42 Aminopeptidases Erroneously Annotated as Cellulases

Dutoit¹,

Brandt²,

Legrain³

et al. 2012

PLoS ONE

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“…However, MetAP lacks the N-terminal domain. In enzyme assays, YpdF was found to have weak activity on substrates with an N-terminal methionine (Met-Xaa) if the second amino acid is alanine, proline, or serine and higher activity for other substrates with a proline in the second position (Xaa-Pro) (21). In E. coli, YpdF is encoded in an operon with YpdE, which is a methionine aminopeptidase.…”

Section: Resultsmentioning

confidence: 99%

“…In E. coli, YpdF is encoded in an operon with YpdE, which is a methionine aminopeptidase. YpdF and YpdE are proposed to work in concert to degrade proteins, with YpdE removing amino acids from the N terminus until a proline is encountered in the second position and YpdF removing the block (21). The M. tuberculosis pepQ product also has homology (ϳ30% amino acid identity) to other E. coli proline aminopeptidases, the pepP and pepQ products, which have both the catalytic C-terminal domain and a noncatalytic N-terminal domain that plays a role in oligomerization and substrate specificity.…”

Section: Resultsmentioning

confidence: 99%

Mutations in pepQ Confer Low-Level Resistance to Bedaquiline and Clofazimine in Mycobacterium tuberculosis

Almeida

Ioerger

Tyagi

et al. 2016

Antimicrob Agents Chemother

181

142

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fThe novel ATP synthase inhibitor bedaquiline recently received accelerated approval for treatment of multidrug-resistant tuberculosis and is currently being studied as a component of novel treatment-shortening regimens for drug-susceptible and multidrug-resistant tuberculosis. In a limited number of bedaquiline-treated patients reported to date, >4-fold upward shifts in bedaquiline MIC during treatment have been attributed to non-target-based mutations in Rv0678 that putatively increase bedaquiline efflux through the MmpS5-MmpL5 pump. These mutations also confer low-level clofazimine resistance, presumably by a similar mechanism. Here, we describe a new non-target-based determinant of low-level bedaquiline and clofazimine cross-resistance in Mycobacterium tuberculosis: loss-of-function mutations in pepQ (Rv2535c), which corresponds to a putative Xaa-Pro aminopeptidase. pepQ mutants were selected in mice by treatment with clinically relevant doses of bedaquiline, with or without clofazimine, and were shown to have bedaquiline and clofazimine MICs 4 times higher than those for the parental H37Rv strain. Coincubation with efflux inhibitors verapamil and reserpine lowered bedaquiline MICs against both mutant and parent strains to a level below the MIC against H37Rv in the absence of efflux pump inhibitors. However, quantitative PCR (qPCR) revealed no significant differences in expression of Rv0678, mmpS5, or mmpL5 between mutant and parent strains. Complementation of a pepQ mutant with the wild-type gene restored susceptibility, indicating that loss of PepQ function is sufficient for reduced susceptibility both in vitro and in mice. Although the mechanism by which mutations in pepQ confer bedaquiline and clofazimine cross-resistance remains unclear, these results may have clinical implications and warrant further evaluation of clinical isolates with reduced susceptibility to either drug for mutations in this gene. Multidrug-resistant tuberculosis (MDR-TB) is a major threat to global control of tuberculosis (TB). When multidrug resistance is not diagnosed, patients respond poorly to standardized first-line regimens and additional resistance may develop. When MDR-TB is diagnosed, current second-line regimens require prolonged treatment durations and are less effective, more toxic, and far more expensive than first-line therapy (1).The diarylquinoline drug bedaquiline (B) received accelerated approval from the U.S. Food and Drug Administration as part of combination therapy for MDR-TB when other alternatives are not available (2). It is now being studied as a component of novel short-course regimens for MDR as well as drug-susceptible TB (ClinicalTrials.gov identifiers NCT02333799, NCT02193776, NCT02589782, NCT02409290, and NCT02454205 [https://clinicaltrials.gov/]). For new drugs such as bedaquiline, it is essential to define and catalog the mechanisms conferring bacterial resistance in order to design appropriate diagnostic tests (including rapid molecular tests), to better manage the treatment of patients who fail ...

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“…ApepP is ubiquitous and conserved from bacteria to vertebrates (Kulkarni and Deobagkar, 2002;Ersahin et al, 2003Ersahin et al, , 2005Zheng et al, 2005). Mammalian ApepP exists in two forms, cytosolic and membrane bound, and functions in the maturation of active peptides, including hormones (Bradykinin), neuropeptides (Substance P) and neurotransmitters (Medeiros and Turner, 1994;Yoshimoto et al, 1994;Kim et al, 2000).…”

Section: Apepp Regulates β-Catenin Abundancementioning

confidence: 99%

Proteomic analysis reveals APC-dependent post translational modifications and identifies a novel regulator of β-catenin

et al. 2016

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Wnt signaling generates patterns in all embryos, from flies to humans, and controls cell fate, proliferation and metabolic homeostasis. Inappropriate Wnt pathway activation results in diseases, including colorectal cancer. The adenomatous polyposis coli (APC) tumor suppressor gene encodes a multifunctional protein that is an essential regulator of Wnt signaling and cytoskeletal organization. Although progress has been made in defining the role of APC in a normal cellular context, there are still significant gaps in our understanding of APC-dependent cellular function and dysfunction. We expanded the APC-associated protein network using a combination of genetics and a proteomic technique called twodimensional difference gel electrophoresis (2D-DIGE). We show that loss of Drosophila Apc2 causes protein isoform changes reflecting misregulation of post-translational modifications (PTMs), which are not dependent on β-catenin transcriptional activity. Mass spectrometry revealed that proteins involved in metabolic and biosynthetic pathways, protein synthesis and degradation, and cell signaling are affected by Apc2 loss. We demonstrate that changes in phosphorylation partially account for the altered PTMs in APC mutants, suggesting that APC mutants affect other types of PTM. Finally, through this approach Aminopeptidase P was identified as a new regulator of β-catenin abundance in Drosophila embryos. This study provides new perspectives on the cellular effects of APC that might lead to a deeper understanding of its role in development.

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Characterization of Two New Aminopeptidases in Escherichia coli

Cited by 14 publications

References 25 publications

Functional Characterization of Two M42 Aminopeptidases Erroneously Annotated as Cellulases

Functional Characterization of Two M42 Aminopeptidases Erroneously Annotated as Cellulases

Mutations in pepQ Confer Low-Level Resistance to Bedaquiline and Clofazimine in Mycobacterium tuberculosis

Proteomic analysis reveals APC-dependent post translational modifications and identifies a novel regulator of β-catenin

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