PEPA

PEPA is an allosteric modulator of AMPA receptors; binds to the GluA2o and GluA3o LBDs and can be utilized as an indicator of AMPA receptor heterogeneity. IC50 value: Target: AMPAR modulator in vitro: PEPA dose-dependently potentiated AMPA-induced increase of [Ca2+]i. In 90% (72 out of 80) of the cells in which cyclothiazide acts, PEPA potentiated the increased [Ca2+]i induced by AMPA with pronounced cell-to-cell variation in rat hippocampal cultures [1]. PEPA bound to the binding domains of the GluA2 and GluA3 flop isoforms of AMPA receptors [2]. coapplication of AMPA with PEPA protected hippocampal CA1 neurons from brain ischemia-induced death. Coapplication of AMPA with PEPA could prevent downregulated expression of GluR2 subunit caused by ischemia and increase BDNF expression via Lyn-ERK1/2-CREB signaling [4]. in vivo: PEPA (3, 10, 30mg/kg body weight) or vehicle was intraperitoneally administered into stressed mice once before the first extinction session. The significant decrease of the freezing response in the extinction sessions was only seen in the 30mg/kg PEPA-administered stressed mice, compared with vehicle-administered stressed mice [3].

References:
[1]. Sekiguchi M, et al. Pharmacological detection of AMPA receptor heterogeneity by use of two allosteric potentiators in rat hippocampal cultures. Br J Pharmacol. 1998 Apr;123(7):1294-303. [2]. Ahmed AH, et al. Molecular mechanism of flop selectivity and subsite recognition for an AMPA receptor allosteric modulator: structures of GluA2 and GluA3 in complexes with PEPA. Biochemistry. 2010 Apr 6;49(13):2843-50. [3]. Yamada D, et al. Facilitating actions of an AMPA receptor potentiator upon extinction of contextually conditioned fear response in stressed mice. Neurosci Lett. 2011 Jan 25;488(3):242-6. [4]. Zhang QG, et al. Positive modulation of AMPA receptors prevents downregulation of GluR2 expression and activates the Lyn-ERK1/2-CREB signaling in rat brain ischemia. Hippocampus. 2010 Jan;20(1):65-77.

Unexpected Diversity of pepA Genes Encoding Leucine Aminopeptidases in Sediments from a Freshwater Lake.[Pubmed:26936797]

Microbes Environ. 2016;31(1):49-55.

We herein designed novel PCR primers for universal detection of the PEPA gene, which encodes the representative leucine aminopeptidase gene, and investigated the genetic characteristics and diversity of PEPA genes in sediments of hypereutrophic Lake Kasumigaura, Japan. Most of the amino acid sequences deduced from the obtained clones (369 out of 370) were related to PEPA-like protein sequences in the M17 family of proteins. The developed primers broadly detected PEPA-like clones associated with diverse bacterial phyla-Alpha-, Beta-, Gamma-, and Deltaproteobacteria, Acidobacteria, Actinobacteria, Aquificae, Chlamydiae, Chloroflexi, Cyanobacteria, Firmicutes, Nitrospirae, Planctomycetes, and Spirochetes as well as the archaeal phylum Thaumarchaeota, indicating that prokaryotes in aquatic environments possessing leucine aminopeptidase are more diverse than previously reported. Moreover, prokaryotes related to the obtained PEPA-like clones appeared to be r- and K-strategists, which was in contrast to our previous findings showing that the neutral metalloprotease gene clones obtained were related to the r-strategist genus Bacillus. Our results suggest that an unprecedented diversity of prokaryotes with a combination of different proteases participate in sedimentary proteolysis.

The Aspergillus nidulans syntaxin PepA(Pep12) is regulated by two Sec1/Munc-18 proteins to mediate fusion events at early endosomes, late endosomes and vacuoles.[Pubmed:26395371]

Mol Microbiol. 2016 Jan;99(1):199-216.

Syntaxins are target-SNAREs that crucially contribute to determine membrane compartment identity. Three syntaxins, Tlg2p, Pep12p and Vam3p, organize the yeast endovacuolar system. Remarkably, filamentous fungi lack the equivalent of the yeast vacuolar syntaxin Vam3p, making unclear how these organisms regulate vacuole fusion. We show that the nearly essential Aspergillus nidulans syntaxin PEPA(Pep12) , present in all endocytic compartments between early endosomes and vacuoles, shares features of Vam3p and Pep12p, and is capable of forming compositional equivalents of all known yeast endovacuolar SNARE bundles including that formed by yeast Vam3p for vacuolar fusion. Our data further indicate that regulation by two Sec1/Munc-18 proteins, Vps45 in early endosomes and Vps33 in early and late endosomes/vacuoles contributes to the wide domain of PEPA(Pep12) action. The syntaxin TlgB(Tlg2) localizing to the TGN appears to mediate retrograde traffic connecting post-Golgi (sorting) endosomes with the TGN. TlgB(Tlg2) is dispensable for growth but becomes essential if the early Golgi syntaxin SedV(Sed5) is compromised, showing that the Golgi can function with a single syntaxin, SedV(Sed5) . Remarkably, its pattern of associations with endosomal SNAREs is consistent with SedV(Sed5) playing roles in retrograde pathway(s) connecting endocytic compartments downstream of the post-Golgi endosome with the Golgi, besides more conventional intra-Golgi roles.

Positional Enrichment by Proton Analysis (PEPA): A One-Dimensional (1) H-NMR Approach for (13) C Stable Isotope Tracer Studies in Metabolomics.[Pubmed:28220994]

Angew Chem Int Ed Engl. 2017 Mar 20;56(13):3531-3535.

A novel metabolomics approach for NMR-based stable isotope tracer studies called PEPA is presented, and its performance validated using human cancer cells. PEPA detects the position of carbon label in isotopically enriched metabolites and quantifies fractional enrichment by indirect determination of (13) C-satellite peaks using 1D-(1) H-NMR spectra. In comparison with (13) C-NMR, TOCSY and HSQC, PEPA improves sensitivity, accelerates the elucidation of (13) C positions in labeled metabolites and the quantification of the percentage of stable isotope enrichment. Altogether, PEPA provides a novel framework for extending the high-throughput of (1) H-NMR metabolic profiling to stable isotope tracing in metabolomics, facilitating and complementing the information derived from 2D-NMR experiments and expanding the range of isotopically enriched metabolites detected in cellular extracts.

The trigger enzyme PepA (aminopeptidase A) of Escherichia coli, a transcriptional repressor that generates positive supercoiling.[Pubmed:27213286]

FEBS Lett. 2016 Jun;590(12):1816-25.

Escherichia coli aminopeptidase A (PEPA) is a trigger enzyme endowed with catalytic activity and DNA-binding properties prominent in transcriptional regulation and site-specific DNA recombination. The current work demonstrates that PEPA is a repressor in its own right, capable of specifically inhibiting transcription initiation at promoter P1 of the carAB operon, encoding carbamoylphosphate synthase. Furthermore, in vitro topology studies performed with DNA minicircles demonstrate that PEPA binding constrains a single positive supercoil in the carP1 control region. Such a topological event is understood to constitute an impediment to transcription initiation and may serve as a mechanism to regulate gene expression.

A desensitization-selective potentiator of AMPA-type glutamate receptors.[Pubmed:12145103]

Br J Pharmacol. 2002 Aug;136(7):1033-41.

1: We examined the effects of PEPA, an allosteric potentiator of AMPA receptors, on AMPA receptor kinetics. 2: PEPA did not affect the deactivation of glutamate responses but potently attenuated the extent of receptor desensitization without slowing the onset of desensitization in most of the recombinant AMPA receptors (GluR1-flip, GluR1-flop, GluR3-flip, GluR3-flip+GluR2-flip, and GluR3-flop+GluR2-flop) expressed in Xenopus oocytes. For the GluR3-flop subunit, PEPA attenuated the extent of desensitization and only weakly prolonged deactivation (1.3 fold). 3: PEPA did not significantly affect recovery from desensitization in oocytes expressing GluR3-flip, GluR1-flop, and GluR1-flop, but weakly accelerated (2.6 fold) recovery from desensitization in oocytes expressing GluR3-flop. 4: PEPA's effect on desensitization of GluR3-flop-containing receptors is unique in that onset is very slow. 5: Simulation studies using simplified kinetic models for AMPA receptors are utilized to explore the differential effects of PEPA on GluR3-flip and -flop. It is possible to simulate the action on GluR3-flip by modulating two rate constants in a 12-state kinetic model. For simulation of the action on GluR3-flop, the 12-state kinetic model is not enough, and it is necessary to invoke a 13th state, a PEPA-bound receptor to which glutamate cannot bind. 6: These results suggest that attenuation of extent of desensitization represents the principal mechanism underlying the potentiation of AMPA receptors by PEPA, and that PEPA exhibits different mechanisms with respect to GluR3-flip and GluR3-flop.

A novel allosteric potentiator of AMPA receptors: 4--2-(phenylsulfonylamino)ethylthio--2,6-difluoro-phenoxyaceta mide.[Pubmed:9221774]

J Neurosci. 1997 Aug 1;17(15):5760-71.

We report that a novel sulfonylamino compound, 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetam ide (PEPA), selectively potentiates glutamate receptors of the AMPA subtype. PEPA (1-200 microM) dose dependently potentiated glutamate-evoked currents in Xenopus oocytes expressing AMPA (GluRA-GluRD), but not kainate (GluR6 and GluR6+KA2) or NMDA (zeta1 + epsilon1-epsilon4), receptor subunits. PEPA was effective at micromolar concentrations and, in contrast to the action of cyclothiazide, preferentially modulated AMPA receptor flop isoforms. At 200 microM, PEPA potentiated glutamate responses by 50-fold in oocytes expressing GluRCflop (EC50 approximately 50 microM) versus only threefold for GluRCflip; a similar preference for flop isoforms was observed for other AMPA receptor subunits. Dose-response analysis for GluRCflop revealed that 100 microM PEPA produced a sevenfold increase in AMPA receptor affinity for glutamate. PEPA produced considerably weaker potentiation of kainate-evoked than glutamate-evoked currents, suggesting modulation of the process of receptor desensitization. In human embryonic kidney 293 cells transfected with AMPA receptor subunits, PEPA either abolished or markedly slowed the rate of onset of desensitization and potentiated steady-state equilibrium currents evoked by glutamate with subunit (GluRC >/= GluRD > GluRA) and splice-variant (flop > flip) selectivity similar to that observed in oocytes. Our results show that PEPA is a novel, flop-preferring allosteric modulator of AMPA receptor desensitization at least 100 times more potent than aniracetam.

PEPA is an allosteric modulator of AMPA receptors; binds to the GluA2o and GluA3o LBDs and can be utilized as an indicator of AMPA receptor heterogeneity.

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