RESUMO
PDZ domain mediated interactions with voltage-gated calcium (Ca V ) channel C-termini play important roles in localizing and compartmentalizing membrane Ca 2+ signaling. The first such interaction discovered was between the neuronal multi-domain protein Mint-1, and the presynaptc calcium channel Ca V 2.2 in mammals. Although the physiological significance of this interaction is unclear, its occurrence in vertebrates and bilaterian invertebrates suggests important and conserved functions. In this study, we explore the evolutionary origins of Mint and its interaction with Ca V 2 channels. Phylogenetic and structural in silico analyses revealed that Mint is an animal-specific gene, like Ca V 2 channels, which bears a highly divergent N-terminus but strongly conserved C-terminus comprised of a phosphotyrosine binding domain, two tandem PDZ domains (PDZ-1 and PDZ-2), and a C-terminal auto-inhibitory element that binds and inhibits PDZ-1. Also deeply conserved are other Mint interacting proteins, namely amyloid precursor and related proteins, presenilins, neurexin, as well as CASK and Veli which form a tripartite complex with Mint in bilaterians. Through yeast 2-hybrid and bacterial 2-hybrid experiments, we show that Mint and Ca V 2 channels from cnidarians and placozoans interact in vitro , and in situ hybridization revealed co-expression of corresponding transcripts in dissociated neurons from the cnidarian Nematostella vectensis . Unexpectedly, the Mint orthologue from the ctenophore Hormiphora californiensis was able to strongly bind the divergent C-terminal ligands of cnidarian and placozoan Ca V 2 channels, despite neither the ctenophore Mint, nor the placozoan and cnidarian orthologues, binding the ctenophore Ca V 2 channel C-terminus. Altogether, our analyses provide a model for the emergence of this interaction in early animals first via adoption of a PDZ ligand by Ca V 2 channels, followed by sequence changes in the ligand that caused a modality switch for binding to Mint.
RESUMO
The Entner-Doudoroff (ED) pathway provides an alternative to glycolysis. It converts 6-phosphogluconate (6-PG) to glyceraldehyde-3-phosphate and pyruvate in two steps consisting of a dehydratase (EDD) and an aldolase (EDA). Here, we investigate its distribution and significance in higher plants and determine the ED pathway is restricted to prokaryotes due to the absence of EDD genes in eukaryotes. EDDs share a common origin with dihydroxy-acid dehydratases (DHADs) of the branched chain amino acid pathway (BCAA). Each dehydratase features strict substrate specificity. E. coli EDD dehydrates 6-PG to 2-keto-3-deoxy-6-phosphogluconate, while DHAD only dehydrates substrates from the BCAA pathway. Structural modeling identifies two divergent domains which account for their non-overlapping substrate affinities. Coupled enzyme assays confirm only EDD participates in the ED pathway. Plastid ancestors lacked EDD but transferred metabolically promiscuous EDA, which explains the absence of the ED pathway from the Viridiplantae and sporadic persistence of EDA genes across the plant kingdom.
Assuntos
Escherichia coli , Via de Pentose Fosfato , Escherichia coli/genética , Glicólise , Ácido Pirúvico , Plantas/metabolismo , Hidroliases/metabolismo , Glucose/metabolismoRESUMO
As the primary enzyme responsible for the activatable conversion of Irinotecan (CPT-11) to SN-38, carboxylesterase 2 (CES2) is a significant predictive biomarker toward CPT-11-based treatments for pancreatic ductal adenocarcinoma (PDAC). High SN-38 levels from high CES2 activity lead to harmful effects, including life-threatening diarrhea. While alternate strategies have been explored, CES2 inhibition presents an effective strategy to directly alter the pharmacokinetics of CPT-11 conversion, ultimately controlling the amount of SN-38 produced. To address this, we conducted a high-throughput screening to discover 18 small-molecule CES2 inhibitors. The inhibitors are validated by dose-response and counter-screening and 16 of these inhibitors demonstrate selectivity for CES2. These 16 inhibitors inhibit CES2 in cells, indicating cell permeability, and they show inhibition of CPT-11 conversion with the purified enzyme. The top five inhibitors prohibited cell death mediated by CPT-11 when preincubated in PDAC cells. Three of these inhibitors displayed a tight-binding mechanism of action with a strong binding affinity.