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1.
Cell ; 176(3): 448-458.e12, 2019 01 24.
Artículo en Inglés | MEDLINE | ID: mdl-30639101

RESUMEN

Cannabis elicits its mood-enhancing and analgesic effects through the cannabinoid receptor 1 (CB1), a G protein-coupled receptor (GPCR) that signals primarily through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Activation of CB1-Gi signaling pathways holds potential for treating a number of neurological disorders and is thus crucial to understand the mechanism of Gi activation by CB1. Here, we present the structure of the CB1-Gi signaling complex bound to the highly potent agonist MDMB-Fubinaca (FUB), a recently emerged illicit synthetic cannabinoid infused in street drugs that have been associated with numerous overdoses and fatalities. The structure illustrates how FUB stabilizes the receptor in an active state to facilitate nucleotide exchange in Gi. The results compose the structural framework to explain CB1 activation by different classes of ligands and provide insights into the G protein coupling and selectivity mechanisms adopted by the receptor.


Asunto(s)
Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB1/ultraestructura , Animales , Agonistas de Receptores de Cannabinoides/farmacología , Cannabinoides/farmacología , Microscopía por Crioelectrón/métodos , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Indazoles/farmacología , Ligandos , Unión Proteica , Receptor Cannabinoide CB1/química , Receptores de Cannabinoides/química , Receptores de Cannabinoides/metabolismo , Receptores de Cannabinoides/ultraestructura , Receptores Acoplados a Proteínas G/metabolismo , Células Sf9 , Transducción de Señal/efectos de los fármacos
2.
Cell ; 173(3): 735-748.e15, 2018 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-29677516

RESUMEN

Teneurins (TENs) are cell-surface adhesion proteins with critical roles in tissue development and axon guidance. Here, we report the 3.1-Å cryoelectron microscopy structure of the human TEN2 extracellular region (ECR), revealing a striking similarity to bacterial Tc-toxins. The ECR includes a large ß barrel that partially encapsulates a C-terminal domain, which emerges to the solvent through an opening in the mid-barrel region. An immunoglobulin (Ig)-like domain seals the bottom of the barrel while a ß propeller is attached in a perpendicular orientation. We further show that an alternatively spliced region within the ß propeller acts as a switch to regulate trans-cellular adhesion of TEN2 to latrophilin (LPHN), a transmembrane receptor known to mediate critical functions in the central nervous system. One splice variant activates trans-cellular signaling in a LPHN-dependent manner, whereas the other induces inhibitory postsynaptic differentiation. These results highlight the unusual structural organization of TENs giving rise to their multifarious functions.


Asunto(s)
Toxinas Bacterianas/química , Proteínas de la Membrana/química , Proteínas del Tejido Nervioso/química , Sinapsis/metabolismo , Empalme Alternativo , Secuencias de Aminoácidos , Animales , Axones , Adhesión Celular , Línea Celular , AMP Cíclico/metabolismo , Femenino , Células HEK293 , Hormonas/química , Humanos , Insectos , Proteínas de la Membrana/metabolismo , Ratones , Conformación Molecular , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuropéptidos/química , Unión Proteica , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/química , Transducción de Señal
3.
Nature ; 604(7907): 757-762, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35418682

RESUMEN

Adhesion G-protein-coupled receptors (aGPCRs) are characterized by the presence of auto-proteolysing extracellular regions that are involved in cell-cell and cell-extracellular matrix interactions1. Self cleavage within the aGPCR auto-proteolysis-inducing (GAIN) domain produces two protomers-N-terminal and C-terminal fragments-that remain non-covalently attached after receptors reach the cell surface1. Upon dissociation of the N-terminal fragment, the C-terminus of the GAIN domain acts as a tethered agonist (TA) peptide to activate the seven-transmembrane domain with a mechanism that has been poorly understood2-5. Here we provide cryo-electron microscopy snapshots of two distinct members of the aGPCR family, GPR56 (also known as ADGRG1) and latrophilin 3 (LPHN3 (also known as ADGRL3)). Low-resolution maps of the receptors in their N-terminal fragment-bound state indicate that the GAIN domain projects flexibly towards the extracellular space, keeping the encrypted TA peptide away from the seven-transmembrane domain. High-resolution structures of GPR56 and LPHN3 in their active, G-protein-coupled states, reveal that after dissociation of the extracellular region, the decrypted TA peptides engage the seven-transmembrane domain core with a notable conservation of interactions that also involve extracellular loop 2. TA binding stabilizes breaks in the middle of transmembrane helices 6 and 7 that facilitate aGPCR coupling and activation of heterotrimeric G proteins. Collectively, these results enable us to propose a general model for aGPCR activation.


Asunto(s)
Receptores Acoplados a Proteínas G , Transducción de Señal , Adhesión Celular , Membrana Celular/metabolismo , Microscopía por Crioelectrón , Humanos , Péptidos/química , Unión Proteica , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos
4.
Nature ; 583(7817): 638-643, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32555463

RESUMEN

N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.


Asunto(s)
Acetilación , Citidina/análogos & derivados , Células Eucariotas/metabolismo , Evolución Molecular , ARN/química , ARN/metabolismo , Archaea/química , Archaea/citología , Archaea/genética , Archaea/crecimiento & desarrollo , Secuencia Conservada , Microscopía por Crioelectrón , Citidina/metabolismo , Células Eucariotas/citología , Células HeLa , Humanos , Modelos Moleculares , Acetiltransferasas N-Terminal/metabolismo , ARN de Archaea/química , ARN de Archaea/genética , Proteínas de Unión al ARN/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Ribosomas/ultraestructura , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Temperatura
5.
Nucleic Acids Res ; 48(20): 11750-11761, 2020 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-33091122

RESUMEN

Ribosomal RNA is the central component of the ribosome, mediating its functional and architectural properties. Here, we report the cryo-EM structure of a highly divergent cytoplasmic ribosome from the single-celled eukaryotic alga Euglena gracilis. The Euglena large ribosomal subunit is distinct in that it contains 14 discrete rRNA fragments that are assembled non-covalently into the canonical ribosome structure. The rRNA is substantially enriched in post-transcriptional modifications that are spread far beyond the catalytic RNA core, contributing to the stabilization of this highly fragmented ribosome species. A unique cluster of five adenosine base methylations is found in an expansion segment adjacent to the protein exit tunnel, such that it is positioned for interaction with the nascent peptide. As well as featuring distinctive rRNA expansion segments, the Euglena ribosome contains four novel ribosomal proteins, localized to the ribosome surface, three of which do not have orthologs in other eukaryotes.


Asunto(s)
Euglena gracilis/química , ARN Ribosómico/química , Ribosomas/química , Microscopía por Crioelectrón , Citoplasma/química , Euglena gracilis/genética , Euglena gracilis/metabolismo , Modelos Moleculares , Procesamiento Postranscripcional del ARN , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/química
6.
Nucleic Acids Res ; 47(5): 2609-2629, 2019 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-30605535

RESUMEN

In trypanosomes, in contrast to most eukaryotes, the large subunit (LSU) ribosomal RNA is fragmented into two large and four small ribosomal RNAs (srRNAs) pieces, and this additional processing likely requires trypanosome-specific factors. Here, we examined the role of 10 abundant small nucleolar RNAs (snoRNAs) involved in rRNA processing. We show that each snoRNA involved in LSU processing associates with factors engaged in either early or late biogenesis steps. Five of these snoRNAs interact with the intervening sequences of rRNA precursor, whereas the others only guide rRNA modifications. The function of the snoRNAs was explored by silencing snoRNAs. The data suggest that the LSU rRNA processing events do not correspond to the order of rRNA transcription, and that srRNAs 2, 4 and 6 which are part of LSU are processed before srRNA1. Interestingly, the 6 snoRNAs that affect srRNA1 processing guide modifications on rRNA positions that span locations from the protein exit tunnel to the srRNA1, suggesting that these modifications may serve as check-points preceding the liberation of srRNA1. This study identifies the highest number of snoRNAs so far described that are involved in rRNA processing and/or rRNA folding and highlights their function in the unique trypanosome rRNA maturation events.


Asunto(s)
Procesamiento Postranscripcional del ARN/genética , ARN Ribosómico/genética , ARN Nuclear Pequeño/genética , Trypanosoma brucei brucei/genética , Conformación de Ácido Nucleico , Precursores del ARN/genética , Transcripción Genética
7.
RNA Biol ; 17(7): 1018-1039, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32250712

RESUMEN

The parasite Trypanosoma brucei cycles between insect and mammalian hosts, and is the causative agent of sleeping sickness. Here, we performed genome-wide mapping of 2'-O-methylations (Nms) on trypanosome rRNA using three high-throughput sequencing methods; RibOxi-seq, RiboMeth-seq and 2'-OMe-seq. This is the first study using three genome-wide mapping approaches on rRNA from the same species showing the discrepancy among the methods. RibOxi-seq detects all the sites, but RiboMeth-seq is the only method to evaluate the level of a single Nm site. The sequencing revealed at least ninety-nine Nms guided by eighty-five snoRNAs among these thirty-eight Nms are trypanosome specific sites. We present the sequence and target of the C/D snoRNAs guiding on rRNA. This is the highest number of Nms detected to date on rRNA of a single cell parasite. Based on RiboMeth-seq, several Nm sites were found to be differentially regulated at the two stages of the parasite life cycle, the insect procyclic form (PCF) versus the bloodstream form (BSF) in the mammalian host.


Asunto(s)
ARN Protozoario , ARN Ribosómico , ARN Nucleolar Pequeño/genética , Trypanosoma brucei brucei/genética , Biología Computacional/métodos , Conectoma , Perfilación de la Expresión Génica , Conformación de Ácido Nucleico , Transcriptoma
8.
Anal Chem ; 91(24): 15634-15643, 2019 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-31725277

RESUMEN

RNA post-transcriptional modifications are common in all kingdoms of life and are predominantly affiliated with methylations at various nucleobase positions. Methylations occur frequently at specific sites on the RNA nucleobases and appear to regulate site-specific intermolecular/intramolecular interactions. Herein, we present a method that utilizes liquid chromatography-mass spectrometry (LC-MS) to identify positional monomethylated RNA nucleoside isomers. The method produces profiles of in-source fragmentation and subsequent tandem mass spectrometry (MS2) (pseudo-MS3) of RNase-digested fragments of an RNA and distinguishes between positional methylated nucleobase isomers by comparing their intranucleobase fragment ion profiles with signature profiles derived from authentic isomers. For method validation, we independently determined the positions of all known monomethylated nucleoside isomers in the Escherichia coli 16S/23S rRNAs. As proof of concept, we further applied this technology to fully characterize the base-modified nucleoside positional isomers, in rRNAs derived from Leishmania donovani, a human blood parasite afflicting millions around the globe. The method described herein will be highly beneficial for the delineation of RNA modification profiles in various cellular RNAs, and as it only requires a subpicomole amount of RNA, it could also be used for the structure-function studies of RNA populations represented in minute amounts in the cell.


Asunto(s)
Escherichia coli/genética , Leishmania/genética , Nucleósidos/análisis , ARN Ribosómico 18S/análisis , ARN Ribosómico/análisis , Humanos , Metilación , Nucleósidos/química , Procesamiento Postranscripcional del ARN , ARN Ribosómico/química , ARN Ribosómico 18S/química
9.
Nucleic Acids Res ; 45(17): 10284-10292, 2017 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-28973455

RESUMEN

Antimicrobial resistance within a wide range of pathogenic bacteria is an increasingly serious threat to global public health. Among these pathogenic bacteria are the highly resistant, versatile and possibly aggressive bacteria, Staphylococcus aureus. Lincosamide antibiotics were proved to be effective against this pathogen. This small, albeit important group of antibiotics is mostly active against Gram-positive bacteria, but also used against selected Gram-negative anaerobes and protozoa. S. aureus resistance to lincosamides can be acquired by modifications and/or mutations in the rRNA and rProteins. Here, we present the crystal structures of the large ribosomal subunit of S. aureus in complex with the lincosamides lincomycin and RB02, a novel semisynthetic derivative and discuss the biochemical aspects of the in vitro potency of various lincosamides. These results allow better understanding of the drugs selectivity as well as the importance of the various chemical moieties of the drug for binding and inhibition.


Asunto(s)
Lincosamidas/farmacología , Subunidades Ribosómicas Grandes Bacterianas/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Benzamidas/química , Benzamidas/farmacología , Sitios de Unión , Clindamicina/química , Clindamicina/farmacología , Cristalización , Cristalografía por Rayos X , Farmacorresistencia Microbiana , Galactósidos/química , Galactósidos/farmacología , Enlace de Hidrógeno , Lincomicina/química , Lincomicina/farmacología , Lincosamidas/química , Estructura Molecular , Subunidades Ribosómicas Grandes Bacterianas/ultraestructura , Staphylococcus aureus/ultraestructura , Electricidad Estática , Relación Estructura-Actividad
10.
Sci Adv ; 9(26): eadf5799, 2023 06 30.
Artículo en Inglés | MEDLINE | ID: mdl-37390210

RESUMEN

Bacterial lipoproteins (BLPs) decorate the surface of membranes in the cell envelope. They function in membrane assembly and stability, as enzymes, and in transport. The final enzyme in the BLP synthesis pathway is the apolipoprotein N-acyltransferase, Lnt, which is proposed to act by a ping-pong mechanism. Here, we use x-ray crystallography and cryo-electron microscopy to chart the structural changes undergone during the progress of the enzyme through the reaction. We identify a single active site that has evolved to bind, individually and sequentially, substrates that satisfy structural and chemical criteria to position reactive parts next to the catalytic triad for reaction. This study validates the ping-pong mechanism, explains the molecular bases for Lnt's substrate promiscuity, and should facilitate the design of antibiotics with minimal off-target effects.


Asunto(s)
Aciltransferasas , Pared Celular , Microscopía por Crioelectrón , Membrana Celular , Lipoproteínas
11.
Biomedicines ; 11(9)2023 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-37760981

RESUMEN

Assessment of structure-activity relationships for anti-protozoan activity revealed a strategy for preparing potent anisomycin derivatives with reduced host toxicity. Thirteen anisomycin analogs were synthesized by modifying the alcohol, amine, and aromatic functional groups. Examination of anti-protozoal activity against various strains of Leishmania and cytotoxicity against leucocytes with comparison against the parent natural product demonstrated typical losses of activity with modifications of the alcohol, amine, and aromatic meta-positions. On the other hand, the para-phenol moiety of anisomycin proved an effective location for introducing substituents without significant loss of anti-protozoan potency. An entry point for differentiating activity against Leishmania versus host has been uncovered by this systematic study.

12.
Nat Struct Mol Biol ; 29(6): 592-603, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35710843

RESUMEN

Many organisms sense light using rhodopsins, photoreceptive proteins containing a retinal chromophore. Here we report the discovery, structure and biophysical characterization of bestrhodopsins, a microbial rhodopsin subfamily from marine unicellular algae, in which one rhodopsin domain of eight transmembrane helices or, more often, two such domains in tandem, are C-terminally fused to a bestrophin channel. Cryo-EM analysis of a rhodopsin-rhodopsin-bestrophin fusion revealed that it forms a pentameric megacomplex (~700 kDa) with five rhodopsin pseudodimers surrounding the channel in the center. Bestrhodopsins are metastable and undergo photoconversion between red- and green-absorbing or green- and UVA-absorbing forms in the different variants. The retinal chromophore, in a unique binding pocket, photoisomerizes from all-trans to 11-cis form. Heterologously expressed bestrhodopsin behaves as a light-modulated anion channel.


Asunto(s)
Canales Iónicos , Rodopsina , Bestrofinas , Rodopsina/química
13.
Science ; 372(6544): 808-814, 2021 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-33858992

RESUMEN

Obesity is a global epidemic that causes morbidity and impaired quality of life. The melanocortin receptor 4 (MC4R) is at the crux of appetite, energy homeostasis, and body-weight control in the central nervous system and is a prime target for anti-obesity drugs. Here, we present the cryo-electron microscopy (cryo-EM) structure of the human MC4R-Gs signaling complex bound to the agonist setmelanotide, a cyclic peptide recently approved for the treatment of obesity. The work reveals the mechanism of MC4R activation, highlighting a molecular switch that initiates satiation signaling. In addition, our findings indicate that calcium (Ca2+) is required for agonist, but not antagonist, efficacy. These results fill a gap in the understanding of MC4R activation and could guide the design of future weight-management drugs.


Asunto(s)
Fármacos Antiobesidad/química , Receptor de Melanocortina Tipo 4/agonistas , Receptor de Melanocortina Tipo 4/química , Saciedad , alfa-MSH/análogos & derivados , Fármacos Antiobesidad/farmacología , Apetito , Sitios de Unión , Calcio/química , Calcio/fisiología , Microscopía por Crioelectrón , Diseño de Fármacos , Células HEK293 , Humanos , Ligandos , Mutación , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Conformación Proteica en Hélice alfa , Dominios Proteicos , Receptor de Melanocortina Tipo 4/genética , Transducción de Señal , alfa-MSH/química , alfa-MSH/farmacología
14.
Nat Commun ; 8(1): 1589, 2017 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-29150609

RESUMEN

Leishmania is a single-celled eukaryotic parasite afflicting millions of humans worldwide, with current therapies limited to a poor selection of drugs that mostly target elements in the parasite's cell envelope. Here we determined the atomic resolution electron cryo-microscopy (cryo-EM) structure of the Leishmania ribosome in complex with paromomycin (PAR), a highly potent compound recently approved for treatment of the fatal visceral leishmaniasis (VL). The structure reveals the mechanism by which the drug induces its deleterious effects on the parasite. We further show that PAR interferes with several aspects of cytosolic translation, thus highlighting the cytosolic rather than the mitochondrial ribosome as the primary drug target. The results also highlight unique as well as conserved elements in the PAR-binding pocket that can serve as hotspots for the development of novel therapeutics.


Asunto(s)
Leishmania/metabolismo , Paromomicina/metabolismo , Ribosomas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Microscopía por Crioelectrón , Citosol/efectos de los fármacos , Citosol/metabolismo , Humanos , Leishmania/genética , Leishmania/ultraestructura , Modelos Moleculares , Paromomicina/química , Paromomicina/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Ribosomas/química , Ribosomas/ultraestructura , Homología de Secuencia de Aminoácido
15.
Cell Rep ; 16(2): 288-294, 2016 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-27373148

RESUMEN

Leishmania is a single-cell eukaryotic parasite of the Trypanosomatidae family, whose members cause an array of tropical diseases. The often fatal outcome of infections, lack of effective vaccines, limited selection of therapeutic drugs, and emerging resistant strains, underline the need to develop strategies to combat these pathogens. The Trypanosomatid ribosome has recently been highlighted as a promising therapeutic target due to structural features that are distinct from other eukaryotes. Here, we present the 2.8-Å resolution structure of the Leishmania donovani large ribosomal subunit (LSU) derived from a cryo-EM map, further enabling the structural observation of eukaryotic rRNA modifications that play a significant role in ribosome assembly and function. The structure illustrates the unique fragmented nature of leishmanial LSU rRNA and highlights the irregular distribution of rRNA modifications in Leishmania, a characteristic with implications for anti-parasitic drug development.


Asunto(s)
Leishmania donovani , Subunidades Ribosómicas Grandes/química , Microscopía por Crioelectrón , Modelos Moleculares , Conformación de Ácido Nucleico , Estructura Cuaternaria de Proteína , Proteínas Protozoarias/química , ARN Protozoario/química , ARN Ribosómico/química , Proteínas Ribosómicas/química , Subunidades Ribosómicas Grandes/ultraestructura
16.
Antibiotics (Basel) ; 5(3)2016 Jun 29.
Artículo en Inglés | MEDLINE | ID: mdl-27367739

RESUMEN

Most ribosomal antibiotics obstruct distinct ribosomal functions. In selected cases, in addition to paralyzing vital ribosomal tasks, some ribosomal antibiotics are involved in cellular regulation. Owing to the global rapid increase in the appearance of multi-drug resistance in pathogenic bacterial strains, and to the extremely slow progress in developing new antibiotics worldwide, it seems that, in addition to the traditional attempts at improving current antibiotics and the intensive screening for additional natural compounds, this field should undergo substantial conceptual revision. Here, we highlight several contemporary issues, including challenging the common preference of broad-range antibiotics; the marginal attention to alterations in the microbiome population resulting from antibiotics usage, and the insufficient awareness of ecological and environmental aspects of antibiotics usage. We also highlight recent advances in the identification of species-specific structural motifs that may be exploited for the design and the creation of novel, environmental friendly, degradable, antibiotic types, with a better distinction between pathogens and useful bacterial species in the microbiome. Thus, these studies are leading towards the design of "pathogen-specific antibiotics," in contrast to the current preference of broad range antibiotics, partially because it requires significant efforts in speeding up the discovery of the unique species motifs as well as the clinical pathogen identification.

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