Biology subjects

Aye, Y.

Publications and source records attributed to Aye, Y..

8 recordsLinked to original sources

bioRxiv2026

Localis-REX: a general protocol for time-resolved spatiofunctional profiling in live cells and C. elegans

We describe Localis-REX, a method for identifying organ or subcellular locale-specific innate reactive electrophilic metabolite (REM) responsive proteins in live Caenorhabdtis elegans and cultured cells. Localis-REX mimics the natural spatiotemporal production of REMs by expressing HaloTag transiently or stably in a specific organ or tissue of live worms or in a designated organelle or subcellular compartment in cultured cells. Following treatment with a submicromolar photocaged REM and washout, light-triggered uncaging generates brief, localized REM pulses near Halo. REM responsive proteins within the vicinity compete to capture the liberated REM before endogenous clearance processes, including glutathione adduction and redox metabolism, enabling selective identification of the most REM-responsive targets under minimally perturned conditions. Localis-REX can be coupled with 1) proteomics to map chemically-actionable local proteomes or 2) live cell and in vivo functional readouts to define biological responses to locale-specific REM-accumulation, such as translation efficiency monitoring through genetic code expansion. The workflow requires 3-6 h, with downstream data acquisition and analysis requiring an additional 24-48 h, depending on the application 1) or 2. We further describe integration of in vivo Localis-REX with ultraID spatial profiling in live C. elegans, enabling expression normalization across profiled locales. Mechanistic validation of newly-identified responder and REM pairs is facilitated using established T-REX and Z-REX target engagement and on-target REM signaling interrogation protocols. Notably, the workflow was initially established as part of a masters thesis project by a student co-author with no prior experience in C. elegans or cell culture research by the master student, highlighting its accessibility to well-trained junior researchers pursuing interdisciplinary studies.

systems biology↗

bioRxiv2024

Organ-specific electrophile responsivity mappingin live C. elegans

Proximity labeling technologies are limited to indexing localized protein residents. Such data-- although valuable--cannot inform on small-molecule responsivity of local residents. We here bridge this gap by demonstrating in live C. elegans how electrophile-sensing propensity in specific organs can be quantitatively mapped and ranked. Using this method, >70% of tissue-specific responders exhibit novel responsivity, independent of tissue-specific abundance. One responder, cyp-33e1--for which both human and worm orthologs are electrophile responsive--marshals stress-dependent gut functions, despite manifesting uniform abundance across all tissues studied. Cyp-33e1s localized electrophile responsivity operates site-specifically, triggering multifaceted responses: electrophile sensing through the catalytic-site cysteine results in partitioning between enzyme inhibition and localized production of a critical metabolite that governs global lipid availability, whereas a rapid dual-cysteine site-specific sensing modulates gut homeostasis. Beyond pinpointing chemical actionability within local proteomes, tissue-specific electrophile responsivity mapping illuminates otherwise intractable locale-specific metabolite signaling and stress response programs influencing organ-specific decision-making. IN BRIEFContext-specific protein reactivity is a cornerstone of biological stress responsivity, and thus has important ramifications for drug discovery; no current method can inform on these parameters. Here we debut a method that cartographs locale-specific actionability to reactive metabolites; Cyp-33e1, a gut-specific responder, emerged to partition between electrophile-driven localized enzymatic turnover that triggers gut-specific metabolite production shaping global lipid storage, and localized electrophile sensing that marshals global stress response. HIGHLIGHTSO_LILocalis-REX maps organ-specific electrophile-responsive proteins in worms C_LIO_LIHits are neither identified by organ-specific Ultra-ID nor biased by localized expression C_LIO_LIHits are enriched in proteins functionally relevant to stress-related phenotypes C_LIO_LILocalized responsivity of gut-specific hit, cyp-33e1, shapes global lipid availability C_LI

molecular biology↗

bioRxiv2024

NCBP1 electrophilic-stress signaling in the nucleus promotes alternatively-spliced S6K1 that dominantly inhibits global translation

Subcellular stress profoundly influences protein synthesis. However, both the nature of spatiotemporally restricted chemical cues and local protein responders to these cues remain elusive. Unlocking these mechanisms requires the ability to functionally map in living systems locale-specific stress-responder proteins and interrogate how chemical modification of each responder impacts proteome synthesis. We resolved this problem by integrating precision localized electrophile generation and genetic code expansion tools. Upon examination of four distinct subcellular locales, only nuclear-targeted electrophile stress stalled translation. We discovered that NCBP1--a nuclear-resident protein with multifaceted roles in eukaryotic mRNA-biogenesis--propagated this nuclear stress signal through a single cysteine (C436) from among its 19 conserved cysteines. This NCBP1(C436)-specific modification elicited alternative splicing of >250 genes. Mechanistically, global protein-synthesis stall was choreographed by impaired association between electrophile-modified NCBP1(C436) and SF3A1, an essential component of spliceosome, triggering the production of alternatively-spliced S6-kinase, whose expression was sufficient to dominantly inhibit protein translation. SummaryNuclear reactive electrophile stress suppresses translation via an unprecedented gain of function mechanism. First NCBP1 is labeled at one of its 19 cysteines. This triggers alternative S6K1 splicing, which is dominant negative for translation.

biochemistry↗

bioRxiv2022

Z-REX uncovers a bifurcation in function of Keap1 paralogs

Studying electrophile signaling is marred by difficulties in parsing changes in pathway flux attributable to on-target, vis-a-vis off-target, modifications. By combining bolus dosing, knockdown, and Z-REX--a tool investigating on-target/on-pathway electrophile signaling, we document that electrophile labeling of one zebrafish-Keap1-paralog (zKeap1b) stimulates Nrf2-driven antioxidant response (AR) signaling (like the human-ortholog). Conversely, zKeap1a is a dominant-negative regulator of electrophile-promoted Nrf2-signaling, and itself is nonpermissive for electrophile-induced Nrf2-upregulation. This behavior is recapitulated in human cells, wherein following electrophile treatment: (1) zKeap1b-transfected cells are permissive for augmented AR-signaling through reduced zKeap1b-Nrf2 binding; (2) zKeap1a-transfected cells are non-permissive for AR-upregulation, as zKeap1a-Nrf2 binding capacity remains unaltered; (3) 1:1 ZKeap1a:zKeap1b-transfected cells show no Nrf2-release from the Keap1-complex, rendering these cells unable to upregulate AR. We identified a zKeap1a-specific point-mutation (C273I) responsible for zKeap1as behavior. Human-Keap1(C273I), of known diminished Nrf2-regulatory capacity, dominantly muted electrophile-induced Nrf2-signaling. These studies highlight divergent and interdependent electrophile signaling behaviors, despite conserved electrophile sensing. Impact statementHow electrophile-sensing versus -signaling marshal stress responses: two zebrafish Keap1-paralogs are equally adept at electrophile-sensing but manifest divergent and co-regulatory electrophile-signaling behaviors.

pharmacology and toxicology↗

bioRxiv2022

Z-REX: Shepherding Reactive Electrophiles to Specific Proteins Expressed either Tissue-Specifically or Ubiquitously, and Recording the Resultant Functional Electrophile-Induced Redox Responses in Larval Fish

Summary of the Protocol ExtensionThis Protocol Extension describes the adaptation of an existing Nature Protocol detailing the use of T-REX (targetable reactive electrophiles and oxidants)--an on-demand redox targeting toolset in cultured cells. The adaptation described here is for use of REX technologies in live zebrafish embryos (Z-REX). Zebrafish embryos expressing a Halo-tagged protein of interest (POI)--either ubiquitously or tissue-specifically--are treated with a HaloTag-specific small-molecule probe housing a photocaged reactive electrophile (either natural electrophiles or synthetic electrophilic drug-like fragments). The reactive electrophile is then photouncaged at a user-defined time, enabling proximity-assisted electrophile-modification of a POI. Functional and phenotypic ramifications of POI-specific modification can then be monitored, by coupling to standard downstream assays, such as, Click chemistry-based POI-labeling and target-occupancy quantification; immunofluorescence or live imaging; RNA-Seq and qRT-PCR analyses of downstream-transcript modulations. Transient expression of requisite Halo-POI in zebrafish embryos is achieved by mRNA injection. Procedures associated with generation of transgenic zebrafish expressing a tissue-specific Halo-POI are also described. The Z-REX experiments can be completed in <1-week using standard techniques. To successfully execute Z-REX, researchers should have basic skills in fish husbandry, imaging, and pathway analysis. Experience with protein or proteome manipulation is useful. This protocol extension is aimed at helping chemical biologists study precision redox events in a model organism and fish biologists perform redox chemical biology.

biochemistry↗

bioRxiv2020

The mRNA-binding protein HuR is a kinetically-privileged electrophile sensor

The key mRNA-binding proteins HuR and AUF1 are reported stress sensors in mammals. Intrigued by recent reports of sensitivity of these proteins to the electrophilic lipid prostaglandin A2 and other redox signals, we here examined their sensing abilities to a prototypical redox-linked lipid-derived electrophile, 4-hydroxynonenal (HNE). Leveraging our T-REX electrophile delivery platform, we found that only HuR, and not AUF1, is a kinetically-privileged sensor of HNE in HEK293T cells, and sensing functions through a specific cysteine, C13. Cells depleted of HuR, upon treatment with HNE, manifest unique alterations in cell viability and Nrf2-transcription-factor-driven antioxidant response (AR), which our recent work shows is regulated by HuR at the Nrf2-mRNA level. Mutagenesis studies showed that C13-specific sensing alone is not sufficient to explain HuR-dependent stress responsivities, further highlighting a complex context-dependent layer of Nrf2/AR regulation through HuR.

biochemistry↗

bioRxiv2019

Clofarabine commandeers the RNR-alpha-ZRANB3 nuclear signaling axis

Ribonucleotide reductase (RNR) is an essential enzyme in DNA-biogenesis and a target of several chemotherapeutics. Here we investigate how anti-leukemic drugs [e.g., clofarabine (ClF)] that target one of the two subunits of RNR, RNR-, affect non-canonical RNR- functions. We discovered that these clinically-approved RNR-inhibiting dATP-analogs inhibit growth by also targeting ZRANB3--a newly-identified DNA-synthesis promoter and nuclear-localized interactor of RNR-. Remarkably, in early time points following drug treatment, ZRANB3-targeting accounted for most of the drug-induced DNA-synthesis suppression and multiple cell types featuring ZRANB3-knockout/knockdown were resistant to these drugs. Additionally, ZRANB3 plays a major role in regulating tumor-invasion and H-rasG12V-promoted transformation in a manner dependent on the recently-discovered interactome of RNR- involving select cytosolic-/nuclear-localized protein-players. The H-rasG12V-promoted transformation--which we show requires ZRANB3-supported DNA-synthesis--was efficiently suppressed by ClF. Such overlooked mechanisms-of-action of approved drugs and a new example of non-oncogene addiction, which is suppressed by RNR-, may advance cancer interventions.

biochemistry↗

bioRxiv2019

Post-transcriptional regulation of Nrf2-mRNA by the mRNA-binding proteins HuR and AUF1

The Nrf2-driven antioxidant response (AR) is a target of covalent drugs and bioactive native electrophiles. However, much of our understanding of AR has centered on protein-level regulation of Nrf2. Here we report a post-transcriptional modality to directly regulate Nrf2-mRNA. Our initial studies focused on the effects of the key mRNA-binding protein (mRBP) HuR on global transcriptomic changes incurred upon oxidant or electrophile stimulation. These data led us to discover a novel role of HuR in regulating Nrf2/AR, and in the process we further identified the related mRBP AUF1 as an additional novel Nrf2/AR regulator. Both mRBPs regulate AR by direct interaction with the Nrf2 transcript. Our data showed that HuR enhances Nrf2-mRNA maturation and promotes its nuclear export; whereas AUF1 stabilizes Nrf2-mRNA. Both mRBPs target the 3'-UTR of Nrf2-mRNA. Using an AR-reporter zebrafish strain, we document that this post-transcriptional control of AR is conserved at the whole-vertebrate level.

biochemistry↗