RESUMO
Polar auxin transport is unique to plants and coordinates their growth and development1,2. The PIN-FORMED (PIN) auxin transporters exhibit highly asymmetrical localizations at the plasma membrane and drive polar auxin transport3,4; however, their structures and transport mechanisms remain largely unknown. Here, we report three inward-facing conformation structures of Arabidopsis thaliana PIN1: the apo state, bound to the natural auxin indole-3-acetic acid (IAA), and in complex with the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). The transmembrane domain of PIN1 shares a conserved NhaA fold5. In the substrate-bound structure, IAA is coordinated by both hydrophobic stacking and hydrogen bonding. NPA competes with IAA for the same site at the intracellular pocket, but with a much higher affinity. These findings inform our understanding of the substrate recognition and transport mechanisms of PINs and set up a framework for future research on directional auxin transport, one of the most crucial processes underlying plant development.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ácidos Indolacéticos , Proteínas de Membrana Transportadoras , Apoproteínas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ácidos Indolacéticos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ftalimidas/metabolismo , Conformação Proteica , Especificidade por SubstratoRESUMO
Indole-3-propionic acid (IPA) is known to be a microbe-derived compound with a similar structure to the phytohormone auxin (indole-3-acetic acid, IAA). Previous studies reported that IPA exhibited auxin-like bioactivities in plants. However, the underlying molecular mechanism is not totally understood. Here, we revealed that IPA modulated lateral root (LR) development via auxin signaling in the model plant Arabidopsis thaliana. Genetic analysis indicated that deficiency of the TIR1/AFB-Aux/IAA-ARF auxin signaling pathway abolished the effects of IPA on regulating LR development. Further biochemical, transcriptomic profiling and cell biological analyses revealed that IPA directly bound to the TIR1/AFB-Aux/IAA coreceptor complex and thus activated downstream gene expression. Therefore, our work revealed that IPA is a potential signaling molecule that modulates plant growth and development by targeting the TIR1/AFB-Aux/IAA-mediated auxin signaling pathway, providing potential insights into root growth regulation in plants.
RESUMO
The transient receptor potential channel subfamily A member 1 (TRPA1) ion channel is an evolutionary conserved polymodal sensor responding to noxious temperature or chemical stimuli. Notably, the thermosensitivity of TRPA1 varies among different species or even different isoforms in the same species. However, the underlying molecular basis of its thermo-gating remains largely unknown. Here, we determine the structures of a heat-sensitive isoform of TRPA1 in Drosophila melanogaster in two distinct conformations with cryo-samples prepared at 8 °C. Large conformational changes are observed in the ankyrin repeat domain (ARD) and the coiled-coil domain between the two states. Remarkably, all 17 ankyrin repeats are mapped in the newly resolved conformation, forming a propeller-like architecture. Two intersubunit interfaces are identified in the amino (N)-terminal domain, and play vital roles during both heat and chemical activation as shown by electrophysiological analysis. With cryo-samples prepared at 35 °C, only one conformation is resolved, suggesting possible state transitions during heat responses. These findings provide a basis for further understanding how the ARD regulates channel functions, and insights into the gating mechanism of TRPA1.
RESUMO
The phytohormone auxin plays central roles in many growth and developmental processes in plants. Development of chemical tools targeting the auxin pathway is useful for both plant biology and agriculture. Here we reveal that naproxen, a synthetic compound with anti-inflammatory activity in humans, acts as an auxin transport inhibitor targeting PIN-FORMED (PIN) transporters in plants. Physiological experiments indicate that exogenous naproxen treatment affects pleiotropic auxin-regulated developmental processes. Additional cellular and biochemical evidence indicates that naproxen suppresses auxin transport, specifically PIN-mediated auxin efflux. Moreover, biochemical and structural analyses confirm that naproxen binds directly to PIN1 protein via the same binding cavity as the indole-3-acetic acid substrate. Thus, by combining cellular, biochemical, and structural approaches, this study clearly establishes that naproxen is a PIN inhibitor and elucidates the underlying mechanisms. Further use of this compound may advance our understanding of the molecular mechanisms of PIN-mediated auxin transport and expand our toolkit in auxin biology and agriculture.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Humanos , Arabidopsis/metabolismo , Naproxeno/farmacologia , Naproxeno/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Ácidos Indolacéticos/metabolismo , Plantas/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/metabolismoRESUMO
In mitosis, accurate chromosome segregation depends on kinetochores that connect centromeric chromatin to spindle microtubules. The centromeres of budding yeast, which are relatively simple, are connected to individual microtubules via a kinetochore constitutive centromere associated network (CCAN). However, the complex centromeres of human chromosomes comprise millions of DNA base pairs and attach to multiple microtubules. Here, by use of cryo-electron microscopy and functional analyses, we reveal the molecular basis of how human CCAN interacts with duplex DNA and facilitates accurate chromosome segregation. The overall structure relates to the cooperative interactions and interdependency of the constituent sub-complexes of the CCAN. The duplex DNA is topologically entrapped by human CCAN. Further, CENP-N does not bind to the RG-loop of CENP-A but to DNA in the CCAN complex. The DNA binding activity is essential for CENP-LN localization to centromere and chromosome segregation during mitosis. Thus, these analyses provide new insights into mechanisms of action underlying kinetochore assembly and function in mitosis.
RESUMO
Paraquat is one of the most widely used nonselective herbicides and has elicited the emergence of paraquat-resistant weeds. However, the molecular mechanisms of paraquat resistance are not completely understood. Here we report the Arabidopsis gain-of-function mutant pqt15-D with significantly enhanced resistance to paraquat and the corresponding gene PQT15, which encodes the Multidrug and Toxic Extrusion (MATE) transporter DTX6. A point mutation at +932 bp in DTX6 causes a G311E amino acid substitution, enhancing the paraquat resistance of pqt15-D, and overexpression of DTX6/PQT15 in the wild-type plants also results in strong paraquat resistance. Moreover, heterologous expression of DTX6 and DTX6-D in Escherichia coli significantly enhances bacterial resistance to paraquat. Importantly, overexpression of DTX6-D enables Arabidopsis plants to tolerate 4 mM paraquat, a near-commercial application level. DTX6/PQT15 is localized in the plasma membrane and endomembrane, and functions as a paraquat efflux transporter as demonstrated by paraquat efflux assays with isolated protoplasts and bacterial cells. Taken together, our results demonstrate that DTX6/PQT15 is an efflux transporter that confers paraquat resistance by exporting paraquat out of the cytosol. These findings reveal a molecular mechanism of paraquat resistance in higher plants and provide a promising candidate gene for engineering paraquat-resistant crops.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Mutação com Ganho de Função/genética , Resistência a Herbicidas , Paraquat/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Fenótipo , Plantas Geneticamente ModificadasRESUMO
Colitis-associated cancer (CAC) has a close relationship with ulcerative colitis (UC). Therapeutic effect of Schisandrin B (SchB) on UC and CAC remains largely unknown. We investigated the preventative effect of SchB on the dextran sulphate sodium (DSS) model of UC and azoxymethane (AOM)/DSS model of CAC. Furthermore, focal adhesion kinase (FAK) activation and influence on commensal microbiota are important for UC treatment. Impact on FAK activation by SchB in UC development was evaluated in vivo and vitro. We also conducted 16S rRNA sequencing to detect regulation of gut microbiota by SchB. Enhanced protection of intestinal epithelial barrier by SchB through activating FAK contributed to protective effect on colon for the fact that protection of SchB can be reversed by inhibition of FAK phosphorylation. Furthermore, influence on gut microbiota by SchB also played a significant role in UC prevention. Our results revealed that SchB was potent to prevent UC by enhancing protection of intestinal epithelial barrier and influence on gut microbiota, which led to inhibition of CAC. SchB was potential to become a new treatment for UC and prevention of CAC.