RESUMEN
D-amino acid oxidase (DAAO)-catalyzed selective oxidative deamination is a very promising process for synthesizing l-amino acids including l-phosphinothricin (l-PPT, a high-efficiency and broad-spectrum herbicide). However, the wild-type DAAO's low activity toward unnatural substrates like d-phosphinothricin (d-PPT) hampers its application. Herein, a DAAO from Caenorhabditis elegans (CeDAAO) was screened and engineered to improve the catalytic potential on d-PPT. First, we designed a novel growth selection system, taking into account the intricate relationship between the growth of Escherichia coli (E. coli) and the catalytic mechanism of DAAO. The developed system was used for high-throughput screening of gene libraries, resulting in the discovery of a variant (M6) with significantly increased catalytic activity against d-PPT. The variant displays different catalytic properties on substrates with varying hydrophobicity and hydrophilicity. Analysis using Alphafold2 modeling and molecular dynamic simulations showed that the reason for the enhanced activity was the substrate-binding pocket with enlarged size and suitable charge distribution. Further QM/MM calculations revealed that the crucial factor for enhancing activity lies in reducing the initial energy barrier of the reductive half reaction. Finally, a comprehensive binding-model index to predict the enhanced activity of DAAO toward d-PPT, and an enzymatic deracemization approach was developed, enabling the efficient synthesis of l-PPT with remarkable efficiency.
Asunto(s)
Aminobutiratos , Caenorhabditis elegans , D-Aminoácido Oxidasa , Escherichia coli , Ingeniería de Proteínas , D-Aminoácido Oxidasa/metabolismo , D-Aminoácido Oxidasa/genética , D-Aminoácido Oxidasa/química , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/enzimología , Caenorhabditis elegans/genética , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/metabolismo , Ingeniería de Proteínas/métodos , Animales , Aminobutiratos/metabolismo , Aminobutiratos/química , Desaminación , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/químicaRESUMEN
In Caenorhabditis elegans, inter-cellular transport of the small non-coding RNA causing systemic RNAi is mediated by the transmembrane protein SID1, encoded by the sid1 gene in the systemic RNAi defective (sid) loci. SID1 shares structural and sequence similarity with cholesterol uptake protein 1 (CHUP1) and is classified as a member of the ChUP family. Although systemic RNAi is not an evolutionarily conserved process, the sid gene products are found across the animal kingdom, suggesting the existence of other novel gene regulatory mechanisms mediated by small non-coding RNAs. Human homologs of sid gene products-hSIDT1 and hSIDT2-mediate contact-dependent lipophilic small non-coding dsRNA transport. Here, we report the structure of recombinant human SIDT1. We find that the extra-cytosolic domain of hSIDT1 adopts a double jelly roll fold, and the transmembrane domain exists as two modules-a flexible lipid binding domain and a rigid transmembrane domain core. Our structural analyses provide insights into the inherent conformational dynamics within the lipid binding domain in ChUP family members.
Asunto(s)
Proteínas de la Membrana , Animales , Humanos , Secuencia de Aminoácidos , Sitios de Unión , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Lípidos/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos/genética , Interferencia de ARNAsunto(s)
Microscopía por Crioelectrón , Interferencia de ARN , ARN Bicatenario , ARN Bicatenario/metabolismo , ARN Bicatenario/química , Modelos Moleculares , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Conformación ProteicaRESUMEN
The nucleic acid transport properties of the systemic RNAi-defective (SID) 1 family make them attractive targets for developing RNA-based therapeutics and drugs. However, the molecular basis for double-stranded (ds) RNA recognition by SID1 family remains elusive. Here, we report the cryo-EM structures of Caenorhabditis elegans (c) SID1 alone and in complex with dsRNA, both at a resolution of 2.2 Å. The dimeric cSID1 interacts with two dsRNA molecules simultaneously. The dsRNA is located at the interface between ß-strand rich domain (BRD)1 and BRD2 and nearly parallel to the membrane plane. In addition to extensive ionic interactions between basic residues and phosphate backbone, several hydrogen bonds are formed between 2'-hydroxyl group of dsRNA and the contact residues. Additionally, the electrostatic potential surface shows three basic regions are fitted perfectly into three major grooves of dsRNA. These structural characteristics enable cSID1 to bind dsRNA in a sequence-independent manner and to distinguish between DNA and RNA. The cSID1 exhibits no conformational changes upon binding dsRNA, with the exception of a few binding surfaces. Structural mapping of dozens of loss-of-function mutations allows potential interpretation of their diverse functional mechanisms. Our study marks an important step toward mechanistic understanding of the SID1 family-mediated dsRNA uptake.
Asunto(s)
Proteínas de Caenorhabditis elegans , ARN Bicatenario , Animales , Sitios de Unión , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Microscopía por Crioelectrón , Modelos Moleculares , Unión Proteica , ARN Bicatenario/química , ARN Bicatenario/metabolismo , ARN Bicatenario/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Electricidad EstáticaRESUMEN
Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, Caenorhabditis elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1's helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , ARN Bicatenario , Ribonucleasa III , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , ARN Bicatenario/metabolismo , Ribonucleasa III/metabolismo , Ribonucleasa III/química , Ribonucleasa III/genética , Microscopía por Crioelectrón , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/genética , ARN Helicasas/metabolismo , ARN Helicasas/genética , ARN Helicasas/química , Unión Proteica , Adenosina Trifosfato/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/químicaRESUMEN
RNA uptake by cells is critical for RNA-mediated gene interference (RNAi) and RNA-based therapeutics. In Caenorhabditis elegans, RNAi is systemic as a result of SID-1-mediated double-stranded RNA (dsRNA) across cells. Despite the functional importance, the underlying mechanisms of dsRNA internalization by SID-1 remain elusive. Here we describe cryogenic electron microscopy structures of SID-1, SID-1-dsRNA complex and human SID-1 homologs SIDT1 and SIDT2, elucidating the structural basis of dsRNA recognition and import by SID-1. The homodimeric SID-1 homologs share conserved architecture, but only SID-1 possesses the molecular determinants within its extracellular domains for distinguishing dsRNA from single-stranded RNA and DNA. We show that the removal of the long intracellular loop between transmembrane helix 1 and 2 attenuates dsRNA uptake and systemic RNAi in vivo, suggesting a possible endocytic mechanism of SID-1-mediated dsRNA internalization. Our study provides mechanistic insights into dsRNA internalization by SID-1, which may facilitate the development of dsRNA applications based on SID-1.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Microscopía por Crioelectrón , ARN Bicatenario , ARN Bicatenario/metabolismo , ARN Bicatenario/química , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Humanos , Modelos Moleculares , Interferencia de ARN , Proteínas de la MembranaRESUMEN
Many microRNA (miRNA)-guided Argonaute proteins can cleave RNA ('slicing'), even though miRNA-mediated target repression is generally cleavage-independent. Here we use Caenorhabditis elegans to examine the role of catalytic residues of miRNA Argonautes in organismal development. In contrast to previous work, mutations in presumed catalytic residues did not interfere with development when introduced by CRISPR. We find that unwinding and decay of miRNA star strands is weakly defective in the catalytic residue mutants, with the largest effect observed in embryos. Argonaute-Like Gene 2 (ALG-2) is more dependent on catalytic residues for unwinding than ALG-1. The miRNAs that displayed the greatest (albeit minor) dependence on catalytic residues for unwinding tend to form stable duplexes with their star strand, and in some cases, lowering duplex stability alleviates dependence on catalytic residues. While a few miRNA guide strands are reduced in the mutant background, the basis of this is unclear since changes were not dependent on EBAX-1, an effector of Target-Directed miRNA Degradation (TDMD). Overall, this work defines a role for the catalytic residues of miRNA Argonautes in star strand decay; future work should examine whether this role contributes to the selection pressure to conserve catalytic activity of miRNA Argonautes across the metazoan phylogeny.
Asunto(s)
Proteínas Argonautas , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , MicroARNs , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , MicroARNs/metabolismo , MicroARNs/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/química , Estabilidad del ARN/genética , Mutación , Dominio Catalítico/genética , Sistemas CRISPR-Cas , Proteínas de Unión al ARNRESUMEN
Lysosomes are degradation and signalling centres crucial for homeostasis, development and ageing1. To meet diverse cellular demands, lysosomes remodel their morphology and function through constant fusion and fission2,3. Little is known about the molecular basis of fission. Here we identify HPO-27, a conserved HEAT repeat protein, as a lysosome scission factor in Caenorhabditis elegans. Loss of HPO-27 impairs lysosome fission and leads to an excessive tubular network that ultimately collapses. HPO-27 and its human homologue MROH1 are recruited to lysosomes by RAB-7 and enriched at scission sites. Super-resolution imaging, negative-staining electron microscopy and in vitro reconstitution assays reveal that HPO-27 and MROH1 self-assemble to mediate the constriction and scission of lysosomal tubules in worms and mammalian cells, respectively, and assemble to sever supported membrane tubes in vitro. Loss of HPO-27 affects lysosomal morphology, integrity and degradation activity, which impairs animal development and longevity. Thus, HPO-27 and MROH1 act as self-assembling scission factors to maintain lysosomal homeostasis and function.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Lisosomas , Animales , Humanos , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestructura , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/ultraestructura , Homeostasis , Longevidad , Lisosomas/metabolismo , Lisosomas/ultraestructura , Secuencias de Aminoácidos , Microscopía ElectrónicaRESUMEN
Transposable elements are genomic parasites that expand within and spread between genomes1. PIWI proteins control transposon activity, notably in the germline2,3. These proteins recognize their targets through small RNA co-factors named PIWI-interacting RNAs (piRNAs), making piRNA biogenesis a key specificity-determining step in this crucial genome immunity system. Although the processing of piRNA precursors is an essential step in this process, many of the molecular details remain unclear. Here, we identify an endoribonuclease, precursor of 21U RNA 5'-end cleavage holoenzyme (PUCH), that initiates piRNA processing in the nematode Caenorhabditis elegans. Genetic and biochemical studies show that PUCH, a trimer of Schlafen-like-domain proteins (SLFL proteins), executes 5'-end piRNA precursor cleavage. PUCH-mediated processing strictly requires a 7-methyl-G cap (m7G-cap) and a uracil at position three. We also demonstrate how PUCH interacts with PETISCO, a complex that binds to piRNA precursors4, and that this interaction enhances piRNA production in vivo. The identification of PUCH concludes the search for the 5'-end piRNA biogenesis factor in C. elegans and uncovers a type of RNA endonuclease formed by three SLFL proteins. Mammalian Schlafen (SLFN) genes have been associated with immunity5, exposing a molecular link between immune responses in mammals and deeply conserved RNA-based mechanisms that control transposable elements.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Endorribonucleasas , ARN de Interacción con Piwi , Animales , Proteínas Argonautas/metabolismo , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Elementos Transponibles de ADN/genética , Endorribonucleasas/química , Endorribonucleasas/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , ARN de Interacción con Piwi/química , ARN de Interacción con Piwi/genética , ARN de Interacción con Piwi/metabolismo , Análogos de Caperuza de ARN/química , Análogos de Caperuza de ARN/metabolismoRESUMEN
Assembly of the CMG (CDC-45-MCM-2-7-GINS) helicase is the key regulated step during eukaryotic DNA replication initiation. Until now, it was unclear whether metazoa require additional factors that are not present in yeast. In this work, we show that Caenorhabditis elegans DNSN-1, the ortholog of human DONSON, functions during helicase assembly in a complex with MUS-101/TOPBP1. DNSN-1 is required to recruit the GINS complex to chromatin, and a cryo-electron microscopy structure indicates that DNSN-1 positions GINS on the MCM-2-7 helicase motor (comprising the six MCM-2 to MCM-7 proteins), by direct binding of DNSN-1 to GINS and MCM-3, using interfaces that we show are important for initiation and essential for viability. These findings identify DNSN-1 as a missing link in our understanding of DNA replication initiation, suggesting that initiation defects underlie the human disease syndrome that results from DONSON mutations.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Replicación del ADN , Proteínas de Mantenimiento de Minicromosoma , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Microscopía por Crioelectrón , Proteínas de Mantenimiento de Minicromosoma/química , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Dominios ProteicosRESUMEN
The UCS (UNC-45/CRO1/She4p) family of proteins has emerged as chaperones specific for the folding, assembly, and function of myosin. UCS proteins participate in various myosin-dependent cellular processes including myofibril organization and muscle functions, cell differentiation, striated muscle development, cytokinesis, and endocytosis. Mutations in the genes that code for UCS proteins cause serious defects in myosin-dependent cellular processes. UCS proteins that contain an N-terminal tetratricopeptide repeat (TPR) domain are called UNC-45. Vertebrates usually possess two variants of UNC-45, the ubiquitous general-cell UNC-45 (UNC-45A) and the striated muscle UNC-45 (UNC-45B), which is exclusively expressed in skeletal and cardiac muscles. Except for the TPR domain in UNC-45, UCS proteins comprise of several irregular armadillo (ARM) repeats that are organized into a central domain, a neck region, and the canonical C-terminal UCS domain that functions as the chaperoning module. With or without TPR, UCS proteins form linear oligomers that serve as scaffolds that mediate myosin folding, organization into myofibrils, repair, and motility. This chapter reviews emerging functions of these proteins with a focus on UNC-45 as a dedicated chaperone for folding, assembly, and function of myosin at protein and potentially gene levels. Recent experimental evidences strongly support UNC-45 as an absolute regulator of myosin, with each domain of the chaperone playing different but complementary roles during the folding, assembly, and function of myosin, as well as recruiting Hsp90 as a co-chaperone to optimize key steps. It is becoming increasingly clear that UNC-45 also regulates the transcription of several genes involved in myosin-dependent cellular processes.
Asunto(s)
Proteínas de Caenorhabditis elegans , Animales , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Chaperonas Moleculares/metabolismo , Miosinas/genética , Miosinas/química , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismoRESUMEN
The initial step in the sensory transduction pathway underpinning hearing and balance in mammals involves the conversion of force into the gating of a mechanosensory transduction channel1. Despite the profound socioeconomic impacts of hearing disorders and the fundamental biological significance of understanding mechanosensory transduction, the composition, structure and mechanism of the mechanosensory transduction complex have remained poorly characterized. Here we report the single-particle cryo-electron microscopy structure of the native transmembrane channel-like protein 1 (TMC-1) mechanosensory transduction complex isolated from Caenorhabditis elegans. The two-fold symmetric complex is composed of two copies each of the pore-forming TMC-1 subunit, the calcium-binding protein CALM-1 and the transmembrane inner ear protein TMIE. CALM-1 makes extensive contacts with the cytoplasmic face of the TMC-1 subunits, whereas the single-pass TMIE subunits reside on the periphery of the complex, poised like the handles of an accordion. A subset of complexes additionally includes a single arrestin-like protein, arrestin domain protein (ARRD-6), bound to a CALM-1 subunit. Single-particle reconstructions and molecular dynamics simulations show how the mechanosensory transduction complex deforms the membrane bilayer and suggest crucial roles for lipid-protein interactions in the mechanism by which mechanical force is transduced to ion channel gating.
Asunto(s)
Caenorhabditis elegans , Microscopía por Crioelectrón , Canales Iónicos , Mecanotransducción Celular , Animales , Arrestinas/química , Arrestinas/metabolismo , Arrestinas/ultraestructura , Caenorhabditis elegans/química , Caenorhabditis elegans/ultraestructura , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/ultraestructura , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/ultraestructura , Activación del Canal Iónico , Canales Iónicos/química , Canales Iónicos/metabolismo , Canales Iónicos/ultraestructura , LípidosRESUMEN
The nascent polypeptide-associated complex (NAC) interacts with newly synthesized proteins at the ribosomal tunnel exit and competes with the signal recognition particle (SRP) to prevent mistargeting of cytosolic and mitochondrial polypeptides to the endoplasmic reticulum (ER). How NAC antagonizes SRP and how this is overcome by ER targeting signals are unknown. Here, we found that NAC uses two domains with opposing effects to control SRP access. The core globular domain prevented SRP from binding to signal-less ribosomes, whereas a flexibly attached domain transiently captured SRP to permit scanning of nascent chains. The emergence of an ER-targeting signal destabilized NAC's globular domain and facilitated SRP access to the nascent chain. These findings elucidate how NAC hands over the signal sequence to SRP and imparts specificity of protein localization.
Asunto(s)
Retículo Endoplásmico/metabolismo , Chaperonas Moleculares/metabolismo , Señales de Clasificación de Proteína , Partícula de Reconocimiento de Señal/metabolismo , Animales , Sitios de Unión , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Humanos , Modelos Moleculares , Chaperonas Moleculares/química , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , Ribosomas/metabolismo , Partícula de Reconocimiento de Señal/química , Ubiquitina/metabolismoRESUMEN
Neurotransmitter release is mediated by proteins that drive synaptic vesicle fusion with the presynaptic plasma membrane. While soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form the core of the fusion apparatus, additional proteins play key roles in the fusion pathway. Here, we report that the C-terminal amphipathic helix of the mammalian accessory protein, complexin (Cpx), exerts profound effects on membranes, including the formation of pores and the efficient budding and fission of vesicles. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that the membrane remodeling activity of Cpx modulates the structure and stability of recombinant exocytic fusion pores. Cpx had particularly strong effects on pores formed by small numbers of SNAREs. Under these conditions, Cpx increased the current through individual pores 3.5-fold, and increased the open time fraction from roughly 0.1 to 1.0. We propose that the membrane sculpting activity of Cpx contributes to the phospholipid rearrangements that underlie fusion by stabilizing highly curved membrane fusion intermediates.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas del Tejido Nervioso/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Células HEK293 , Humanos , Membrana Dobles de Lípidos/química , Fusión de Membrana/fisiología , Simulación de Dinámica Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Poro Nuclear/química , Poro Nuclear/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica en Hélice alfa , Estabilidad Proteica , Vesículas Sinápticas/química , Vesículas Sinápticas/metabolismoRESUMEN
Aberrant dopamine (DA) signaling is associated with several psychiatric disorders, such as autism, bipolar disorder, addiction, and Parkinson's disease, and several medications that target the DA transporter (DAT) can induce or treat these disorders. In addition, psychostimulants, such as cocaine and D-amphetamine (AMPH), rely on the competitive interactions with the transporter's substrate binding site to produce their rewarding effects. Agents that exhibit noncompetitive, allosteric modulation of DAT remain an important topic of investigation due to their potential therapeutic applications. We previously identified a novel allosteric modulator of human DAT, KM822, that can decrease the affinity of cocaine for DAT and attenuate cocaine-elicited behaviors; however, whether DAT is the sole mediator of KM822 actions in vivo is unproven given the large number of potential off-target sites. Here, we provide in silico and in vitro evidence that the allosteric site engaged by KM822 is conserved between human DAT and Caenorhabditis elegans DAT-1. KM822 binds to a similar pocket in DAT-1 as previously identified in human DAT. In functional dopamine uptake assays, KM822 affects the interaction between AMPH and DAT-1 by reducing the affinity of AMPH for DAT-1. Finally, through a combination of genetic and pharmacological in vivo approaches we provide evidence that KM822 diminishes the behavioral actions of AMPH on swimming-induced paralysis through a direct allosteric modulation of DAT-1. More broadly, our findings demonstrate allosteric modulation of DAT as a behavior modifying strategy and suggests that Caenorhabditis elegans can be operationalized to identify and investigate the interactions of DAT allosteric modulators. SIGNIFICANCE STATEMENT: We previously demonstrated that the dopamine transporter (DAT) allosteric modulator KM822 decreases cocaine affinity for human DAT. Here, using in silico and in vivo genetic approaches, we extend this finding to interactions with amphetamine, demonstrating evolutionary conservation of the DAT allosteric site. In Caenorhabditis elegans, we report that KM822 suppresses amphetamine behavioral effects via specific interactions with DAT-1. Our findings reveal Caenorhabditis elegans as a new tool to study allosteric modulation of DAT and its behavioral consequences.
Asunto(s)
Anfetamina/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Dopaminérgicos/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Anfetamina/farmacología , Animales , Células COS , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Chlorocebus aethiops , Dopaminérgicos/farmacología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/química , Relación Dosis-Respuesta a Droga , Drosophila melanogaster , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Estructura Secundaria de ProteínaRESUMEN
The maintenance of optimal membrane composition under basal and stress conditions is critical for the survival of an organism. High-glucose stress has been shown to perturb membrane properties by decreasing membrane fluidity, and the membrane sensor PAQR-2 is required to restore membrane integrity. However, the mechanisms required to respond to elevated dietary glucose are not fully established. In this study, we used a 13C stable isotope-enriched diet and mass spectrometry to better understand the impact of glucose on fatty acid dynamics in the membrane of Caenorhabditis elegans. We found a novel role for monomethyl branched-chain fatty acids (mmBCFAs) in mediating the ability of the nematodes to survive conditions of elevated dietary glucose. This requirement of mmBCFAs is unique to glucose stress and was not observed when the nematode was fed elevated dietary saturated fatty acid. In addition, when worms deficient in elo-5, the major biosynthesis enzyme of mmBCFAs, were fed Bacillus subtilis (a bacteria strain rich in mmBCFAs) in combination with high glucose, their survival rates were rescued to wild-type levels. Finally, the results suggest that mmBCFAs are part of the PAQR-2 signaling response during glucose stress. Taken together, we have identified a novel role for mmBCFAs in stress response in nematodes and have established these fatty acids as critical for adapting to elevated glucose.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Ácidos Grasos , Glucosa , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Fluidez de la Membrana/fisiología , Proteínas de la MembranaRESUMEN
The emergence of biomolecular condensation and liquid-liquid phase separation (LLPS) introduces a new layer of complexity into our understanding of cell and molecular biology. Evidence steadily grows indicating that condensates are not only implicated in physiology but also human disease. Macro- and mesoscale characterization of condensates as a whole have been instrumental in understanding their biological functions and dysfunctions. By contrast, the molecular level characterization of condensates and how condensates modify the properties of the molecules that constitute them thus far remain comparably scarce. In this minireview we summarize and discuss the findings of several recent studies that have focused on structure, dynamics, and interactions of proteins undergoing condensation. The mechanistic insights they provide help us identify the relevant properties nature and scientists can leverage to modulate the behavior of condensate systems. We also discuss the unique environment of the droplet surface and speculate on effects of topological constraints and physical exclusion on condensate properties.
Asunto(s)
Condensados Biomoleculares/química , Proteínas/química , Condensados Biomoleculares/metabolismo , Fenómenos Biofísicos , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Nucleofosmina/química , Nucleofosmina/metabolismo , Conformación Proteica , Proteínas/metabolismo , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismoRESUMEN
Nuage are RNA-rich condensates that assemble around the nuclei of developing germ cells. Many proteins required for the biogenesis and function of silencing small RNAs (sRNAs) enrich in nuage, and it is often assumed that nuage is the cellular site where sRNAs are synthesized and encounter target transcripts for silencing. Using C. elegans as a model, we examine the complex multicondensate architecture of nuage and review evidence for compartmentalization of silencing pathways. We consider the possibility that nuage condensates balance the activity of competing sRNA pathways and serve to limit, rather than enhance, sRNA amplification to protect transcripts from dangerous runaway silencing.
Asunto(s)
Condensados Biomoleculares/química , Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/química , Interferencia de ARN , ARN de Helminto/química , ARN Interferente Pequeño/química , Animales , Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Condensados Biomoleculares/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Compartimento Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Embrión no Mamífero , Gránulos de Ribonucleoproteína de Células Germinales/metabolismo , Gránulos de Ribonucleoproteína de Células Germinales/ultraestructura , Células Germinativas/metabolismo , Células Germinativas/ultraestructura , ARN de Helminto/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
Complex biological functions within organisms are frequently orchestrated by systemic communication between tissues. In the model organism Caenorhabditis elegans, the pharyngeal and body wall neuromuscular junctions are two discrete structures that control feeding and locomotion, respectively. Separate, the well-defined neuromuscular circuits control these distinct tissues. Nonetheless, the emergent behaviors, feeding and locomotion, are coordinated to guarantee the efficiency of food intake. Here, we show that pharmacological hyperactivation of cholinergic transmission at the body wall muscle reduces the rate of pumping behavior. This was evidenced by a systematic screening of the effect of the cholinesterase inhibitor aldicarb on the rate of pharyngeal pumping on food in mutant worms. The screening revealed that the key determinants of the inhibitory effect of aldicarb on pharyngeal pumping are located at the body wall neuromuscular junction. In fact, the selective stimulation of the body wall muscle receptors with the agonist levamisole inhibited pumping in a lev-1-dependent fashion. Interestingly, this response was independent of unc-38, an alpha subunit of the nicotinic receptor classically expressed with lev-1 at the body wall muscle. This implies an uncharacterized lev-1-containing receptor underpins this effect. Overall, our results reveal that body wall cholinergic transmission not only controls locomotion but simultaneously inhibits feeding behavior.
Asunto(s)
Proteínas de Caenorhabditis elegans , Inhibidores de la Colinesterasa , Conducta Alimentaria , Unión Neuromuscular , Aldicarb/farmacología , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Inhibidores de la Colinesterasa/farmacología , Conducta Alimentaria/efectos de los fármacos , Conducta Alimentaria/fisiología , Levamisol/farmacología , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/metabolismo , Transducción de SeñalRESUMEN
Malate dehydrogenase (MDH) catalyzes the conversion of NAD+ and malate to NADH and oxaloacetate in the citric acid cycle. Eukaryotes have one MDH isozyme that is imported into the mitochondria and one in the cytoplasm. We overexpressed and purified Caenorhabditis elegans cytoplasmic MDH-1 and mitochondrial MDH-2 in E. coli. Our goal was to compare the kinetic and structural properties of these enzymes because C. elegans can survive adverse environmental conditions, such as lack of food and elevated temperatures. In steady-state enzyme kinetics assays, we measured KM values for oxaloacetate of 54 and 52 µM and KM values for NADH of 61 and 107 µM for MDH-1 and MDH-2, respectively. We partially purified endogenous MDH-1 and MDH-2 from a mixed population of worms and separated them using anion exchange chromatography. Both endogenous enzymes had a KM for oxaloacetate similar to that of the corresponding recombinant enzyme. Recombinant MDH-1 and MDH-2 had maximum activity at 40 °C and 35 °C, respectively. In a thermotolerance assay, MDH-1 was much more thermostable than MDH-2. Protein homology modeling predicted that MDH-1 had more intersubunit salt-bridges than mammalian MDH1 enzymes, and these ionic interactions may contribute to its thermostability. In contrast, the MDH-2 homology model predicted fewer intersubunit ionic interactions compared to mammalian MDH2 enzymes. These results suggest that the increased stability of MDH-1 may facilitate its ability to remain active in adverse environmental conditions. In contrast, MDH-2 may use other strategies, such as protein binding partners, to function under similar conditions.