RESUMEN
Rpb1, the largest subunit of RNA polymerase II (RNAPII), is rapidly polyubiquitinated and degraded in response to DNA damage; this process is considered to be a "mechanism of last resort'' employed by cells. The underlying mechanism of this process remains elusive. Here, we uncovered a previously uncharacterized multistep pathway in which the polymerase-associated factor 1 (Paf1) complex (PAF1C, composed of the subunits Ctr9, Paf1, Leo1, Cdc73, and Rtf1) is involved in regulating the RNAPII pool by stimulating Elongin-Cullin E3 ligase complex-mediated Rpb1 polyubiquitination and subsequent degradation by the proteasome following DNA damage. Mechanistically, Spt5 is dephosphorylated following DNA damage, thereby weakening the interaction between the Rtf1 subunit and Spt5, which might be a key step in initiating Rpb1 degradation. Next, Rad26 is loaded onto stalled RNAPII to replace the Spt4/Spt5 complex in an RNAPII-dependent manner and, in turn, recruits more PAF1C to DNA lesions via the binding of Rad26 to the Leo1 subunit. Importantly, the PAF1C, assembled in a Ctr9-mediated manner, coordinates with Rad26 to localize the Elongin-Cullin complex on stalled RNAPII, thereby inducing RNAPII removal, in which the heterodimer Paf1/Leo1 and the subunit Cdc73 play important roles. Together, our results clearly revealed a new role of the intact PAF1C in regulating the RNAPII pool in response to DNA damage.
Asunto(s)
Proteínas Cullin , Daño del ADN , Elonguina , Proteínas Nucleares , ARN Polimerasa II , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Ciclo Celular/metabolismo , Proteínas Cullin/metabolismo , Elonguina/genética , Elonguina/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional/metabolismoRESUMEN
The highly conserved multifunctional polymerase-associated factor 1 (Paf1) complex (PAF1C), composed of five core subunits Paf1, Leo1, Ctr9, Cdc73, and Rtf1, participates in all stages of transcription and is required for the Rad6/Bre1-mediated monoubiquitination of histone H2B (H2Bub). However, the molecular mechanisms underlying the contributions of the PAF1C subunits to H2Bub are not fully understood. Here, we report that Ctr9, acting as a hub, interacts with the carboxyl-terminal acidic tail of Rad6, which is required for PAF1C-induced stimulation of H2Bub. Importantly, we found that the Ras-like domain of Cdc73 has the potential to accelerate ubiquitin discharge from Rad6 and thus facilitates H2Bub, a process that might be conserved from yeast to humans. Moreover, we found that Rtf1 HMD stimulates H2Bub, probably through accelerating ubiquitin discharge from Rad6 alone or in cooperation with Cdc73 and Bre1, and that the Paf1/Leo1 heterodimer in PAF1C specifically recognizes the histone H3 tail of nucleosomal substrates, stimulating H2Bub. Collectively, our biochemical results indicate that intact PAF1C is required to efficiently stimulate Rad6/Bre1-mediated H2Bub.
Asunto(s)
Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Clonación Molecular , Escherichia coli , Regulación Fúngica de la Expresión Génica , Histonas , Proteínas Nucleares/genética , Nucleosomas , Subunidades de Proteína , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismo , Enzimas Ubiquitina-Conjugadoras/genéticaRESUMEN
Self-sorting is a common phenomenon in eukaryotic cells and represents one of the versatile strategies for the formation of advanced functional materials; however, developing artificial self-sorting assemblies within living cells remains challenging. Here, we report on the GSH-responsive in situ self-sorting peptide assemblies within cancer cells for simultaneous organelle targeting to promote combinatorial organelle dysfunction and thereby cell death. The self-sorting system was created via the design of two peptides E3C16E6 and EVMSeO derived from lipid-inspired peptide interdigitating amphiphiles and peptide bola-amphiphiles, respectively. The distinct organization patterns of the two peptides facilitate their GSH-induced self-sorting into isolated nanofibrils as a result of cleavage of disulfide-connected hydrophilic domains or reduction of selenoxide groups. The GSH-responsive in situ self-sorting in the peptide assemblies within HeLa cells was directly characterized by super-resolution structured illumination microscopy. Incorporation of the thiol and ER-targeting groups into the self-sorted assemblies endows their simultaneous targeting of endoplasmic reticulum and Golgi apparatus, thus leading to combinatorial organelle dysfunction and cell death. Our results demonstrate the establishment of the in situ self-sorting peptide assemblies within living cells, thus providing a unique platform for drug targeting delivery and an alternative strategy for modulating biological processes in the future.
Asunto(s)
Aparato de Golgi , Péptidos , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Péptidos/química , Transporte de ProteínasRESUMEN
Enzyme-regulated in situ self-assembly of peptides represents one versatile strategy in the creation of theranostic agents, which, however, is limited by the strong dependence on enzyme overexpression. Herein, we reported the self-amplifying assembly of peptides precisely in macrophages associated with enzyme expression for improving the anti-inflammatory efficacy of conventional drugs. The self-amplifying assembling system was created via coassembling an enzyme-responsive peptide with its derivative functionalized with a protein ligand. Reduction of the peptides by the enzyme NAD(P)H quinone dehydrogenase 1 (NQO1) led to the formation of nanofibers with high affinity to the protein, thereby facilitating NQO1 expression. The improved NQO1 level conversely promoted the assembly of the peptides into nanofibers, thus establishing an amplifying relationship between the peptide assembly and the NQO1 expression in macrophages. Utilization of the amplifying assembling system as vehicles for drug dexamethasone allowed for its passive targeting delivery to acute injured lungs. Both in vitro and in vivo studies confirmed the capability of the self-amplifying assembling system to enhance the anti-inflammatory efficacy of dexamethasone via simultaneous alleviation of the reactive oxygen species side effect and downregulation of proinflammatory cytokines. Our findings demonstrate the manipulation of the assembly of peptides in living cells with a regular enzyme level via a self-amplification process, thus providing a unique strategy for the creation of supramolecular theranostic agents in living cells.
Asunto(s)
Nanofibras , Péptidos , Dexametasona , Ligandos , Macrófagos/metabolismo , Nanofibras/química , Péptidos/químicaRESUMEN
Mitochondrion-targeting therapy exhibits great potential in cancer therapy but significantly suffers from limited therapeutic efficiency. Here we report on mitochondrion-targeting supramolecular antagonist-inducing tumor cell death via simultaneously promoting cellular apoptosis and preventing survival. The supramolecular antagonist was created via coassembly of a mitochondrion-targeting pentapeptide with its two derivatives functionalized with a BH3 domain or the drug camptothecin (CPT). While drug CPT released from the antagonist induced cellular apoptosis via decreasing the mitochondrial membrane potential, the BH3 domain prevented cellular survival through facilitating the association between the supramolecular antagonists and antiapoptotic proteins, thereby initiating mitochondrial permeabilization. Both in vitro and in vivo studies confirmed the combinatorial therapeutic effect arising from the BH3 domain and CPT drug within the supramolecular antagonist on cell death and thereby inhibiting tumor growth. Our findings demonstrate an efficient combinatorial mechanism for mitochondrial dysfunction, thus potentially serving as novel organelle-targeting medicines.
Asunto(s)
Apoptosis , Camptotecina , Camptotecina/farmacología , MitocondriasRESUMEN
NF2/Merlin is an upstream regulator of hippo pathway, and it has two states: an auto-inhibited "closed" state and an active "open" form. Previous studies showed that Drosophila Merlin adopts a more closed conformation. However, the molecular mechanism of conformational regulation remains poorly understood. Here, we first confirmed the strong interaction between FERM and the C-terminal domain (CTD) of Merlin, and then determined the crystal structure of the FERM/CTD complex, which reveals the structural basis of Merlin adopting a more closed conformation compared to its human cognate NF2. Interestingly, we found that the conserved lipid-binding site of Merlin might be masked by a linker. Confocal analyses confirmed that all putative lipid-binding site are very important for the membranal location of Merlin. More, we found that the phosphomimic Thr616Asp mutation weakens the interaction between FERM and CTD of Merlin. Collectively, the crystal structure of the FERM/CTD complex not only provides a mechanistic explanation of functionally dormant conformation of Merlin may also serve as a foundation for revealing the mechanism of conformational regulation of Merlin.
Asunto(s)
Drosophila melanogaster/química , Neurofibromina 2/química , Neurofibromina 2/metabolismo , Multimerización de Proteína , Animales , Sitios de Unión , Drosophila melanogaster/genética , Lípidos , Modelos Moleculares , Mutación , Neurofibromina 2/genética , Fosfoinositido Fosfolipasa C/metabolismo , Unión Proteica , Dominios Proteicos , Multimerización de Proteína/genéticaRESUMEN
The SWI/SNF chromatin remodeling complex, which alters nucleosome positions by either evicting histones or sliding nucleosomes on DNA, is highly conserved from yeast to humans, and 20% of all human cancers have mutations in various subunits of the SWI/SNF complex. Here, we reported the crystal structure of the yeast Snf5-Swi3 subcomplex at a resolution of 2.65 Å. Our results showed that the Snf5-Swi3 subcomplex assembles into a heterotrimer with one Snf5 molecule bound to two distinct Swi3 molecules. In addition, we demonstrated that Snf5-Swi3 subcomplex formation is required for SWI/SNF function in yeast. These findings shed light on the important role of the Snf5-Swi3 subcomplex in the assembly and functional integrity of the SWI/SNF complex.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas de Unión al ADN/química , Células HEK293 , Humanos , Modelos Moleculares , Proteínas Nucleares/química , Unión Proteica , Multimerización de Proteína , Proteínas de Saccharomyces cerevisiae/química , Soluciones , Factores de Transcripción/químicaRESUMEN
Pentatricopeptide repeat (PPR) proteins represent a large family of sequence-specific RNA-binding proteins that are involved in multiple aspects of RNA metabolism. PPR proteins, which are found in exceptionally large numbers in the mitochondria and chloroplasts of terrestrial plants, recognize single-stranded RNA (ssRNA) in a modular fashion. The maize chloroplast protein PPR10 binds to two similar RNA sequences from the ATPI-ATPH and PSAJ-RPL33 intergenic regions, referred to as ATPH and PSAJ, respectively. By protecting the target RNA elements from 5' or 3' exonucleases, PPR10 defines the corresponding 5' and 3' messenger RNA termini. Despite rigorous functional characterizations, the structural basis of sequence-specific ssRNA recognition by PPR proteins remains to be elucidated. Here we report the crystal structures of PPR10 in RNA-free and RNA-bound states at resolutions of 2.85 and 2.45 Å, respectively. In the absence of RNA binding, the nineteen repeats of PPR10 are assembled into a right-handed superhelical spiral. PPR10 forms an antiparallel, intertwined homodimer and exhibits considerable conformational changes upon binding to its target ssRNA, an 18-nucleotide PSAJ element. Six nucleotides of PSAJ are specifically recognized by six corresponding PPR10 repeats following the predicted code. The molecular basis for the specific and modular recognition of RNA bases A, G and U is revealed. The structural elucidation of RNA recognition by PPR proteins provides an important framework for potential biotechnological applications of PPR proteins in RNA-related research areas.
Asunto(s)
Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , ARN/metabolismo , Zea mays/química , Sitios de Unión , Cristalografía por Rayos X , Proteínas de Plantas/genética , Unión Proteica , Estructura Terciaria de Proteína , ARN/química , Zea mays/genética , Zea mays/metabolismoRESUMEN
Liver fibrosis is a common pathological response to chronic hepatic injury. STAT3 is actively involved in the fibrogenesis and angiogenesis seen in liver fibrosis. S3I-201 (NSC 74859) is a chemical inhibitor of STAT3 activity, which blocks the dimerization of STAT3, STAT3-DNA binding and transcription activity. This study evaluated the effects of S3I-201 against liver fibrosis. S3I-201 inhibited the proliferation, migration, and actin filament formation in primary human hepatic stellate cells (HSCs), as well as the expression of α-SMA, collagen I and TIMP1 in both primary HSC and in a CCl4-induced fibrosis mouse model. S3I-201 induced both apoptosis and cell cycle arrest in the HSC cell line (LX-2). S3I-201 inhibited the expression of fibrogenesis factors TGFß1 and TGFßRII, as well as the downstream phosphorylation of Smad2, Smad3, Akt and ERK induced by TGFß1. In addition to fibrogenesis, both in vitro and in vivo assays showed that S3I-201 inhibited angiogenesis through expression suppression of VEGF and VEGFR2. Moreover, S3I-201 also had a synergistic effect with sorafenib, an FDA approved liver cancer drug, in the proliferation, apoptosis, angiogenesis and fibrogenesis of HSC. S3I-201 suppressed liver fibrosis through multiple mechanisms, and combined with sorafenib, S3I-201 could be a potentially effective antifibrotic agent.
Asunto(s)
Bencenosulfonatos/uso terapéutico , Células Estrelladas Hepáticas/efectos de los fármacos , Cirrosis Hepática/prevención & control , Neovascularización Fisiológica/efectos de los fármacos , Factor de Transcripción STAT3/antagonistas & inhibidores , Ácidos Aminosalicílicos/farmacología , Ácidos Aminosalicílicos/uso terapéutico , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Bencenosulfonatos/farmacología , Línea Celular , Sinergismo Farmacológico , Humanos , Masculino , Ratones Endogámicos BALB C , Cultivo Primario de Células , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Sorafenib/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The evolutionarily conserved Elongator complex, which is composed of six subunits elongator protein 1 (Elp1 to -6), plays vital roles in gene regulation. The molecular hallmark of familial dysautonomia (FD) is the splicing mutation of Elp1 [also known as IκB kinase complex-associated protein (IKAP)] in the nervous system that is believed to be the primary cause of the devastating symptoms of this disease. Here, we demonstrate that disease-related mutations in Elp1 affect Elongator assembly, and we have determined the structure of the C-terminal portion of human Elp1 (Elp1-CT), which is sufficient for full-length Elp1 dimerization, as well as the structure of the cognate dimerization domain of yeast Elp1 (yElp1-DD). Our study reveals that the formation of the Elp1 dimer contributes to its stability in vitro and in vivo and is required for the assembly of both the human and yeast Elongator complexes. Functional studies suggest that Elp1 dimerization is essential for yeast viability. Collectively, our results identify the evolutionarily conserved dimerization domain of Elp1 and suggest that the pathological mechanisms underlying the onset and progression of Elp1 mutation-related disease may result from impaired Elongator activities.
Asunto(s)
Proteínas Portadoras/química , Empalme del ARN/genética , Animales , Proteínas Portadoras/genética , Secuencia Conservada , Dimerización , Progresión de la Enfermedad , Disautonomía Familiar/genética , Disautonomía Familiar/fisiopatología , Histona Acetiltransferasas/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos , Factores de Elongación de Péptidos/química , Fenotipo , Conformación Proteica , Mapeo de Interacción de Proteínas , Estabilidad Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Relación Estructura-Actividad , Factores de Elongación TranscripcionalRESUMEN
Merlin functions as a tumor suppressor and suppresses malignant activity of cancer cells through multiple mechanisms. However, whether Serine 518 phosphorylation regulates the conformation of Merlin as well as the open-closed conformational changes affect Merlin's tumor inhibitory activity remain controversial. In this study, we used different mutants to mimic related conformational states of Merlin and investigated its physiological functions. Our results showed that the phosphorylation at Serine 518 has no influence on Merlin's conformation, subcellular localization, or cell proliferation inhibitory activity. As a fully closed conformational state, the A585W mutant loses the ability to recruit Lats2 to the cell membrane, but it does not affect its subcellular distribution or cell proliferation inhibitory activity. As a fully open conformational state, mimicking the conformation of Merlin isoform II, the ΔEL mutant has the same physiological function as the wild type Merlin isoform I. Collectively, we provide for the first time in vivo evidence that the function of Merlin, as a tumor suppressor is independent of its conformational change.
Asunto(s)
Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neurofibromina 2/metabolismo , Neurofibromina 2/ultraestructura , Fosfotransferasas/metabolismo , Serina/metabolismo , Fracciones Subcelulares/metabolismo , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Genes Supresores de Tumor , Humanos , Neoplasias Experimentales/genética , Neurofibromina 2/genética , Fosforilación , Conformación Proteica , Serina/genética , Relación Estructura-ActividadRESUMEN
Pentatricopeptide repeat (PPR) proteins, particularly abundant in plastids and mitochrondria of angiosperms, include a large number of sequence-specific RNA binding proteins that are involved in diverse aspects of organelle RNA metabolisms. PPR proteins contain multiple tandom repeats, and each repeat can specifically recognize a RNA base through residues 2, 5, and 35 in a modular fashion. The crystal structure of PPR10 from maize chloroplast exhibits dimeric existence both in the absence and presence of the 18-nucleotide psaJ RNA element. However, previous biochemical analysis suggested a monomeric shift of PPR10 upon RNA binding. In this report, we show that the amino-terminal segments of PPR10 determine the dimerization state of PPR10. A single amino acid alteration of cysteine to serine within repeat 10 of PPR10 further drives dimerization of PPR10. The biochemical elucidation of the determinants for PPR10 dimerization may provide an important foundation to understand the working mechanisms of PPR proteins underlying their diverse physiological functions.
Asunto(s)
Proteínas de Cloroplastos/química , Proteínas de Plantas/química , Proteínas de Unión al ARN/química , Zea mays/química , Adenina/química , Secuencia de Aminoácidos , Arabidopsis/química , Proteínas de Cloroplastos/genética , Cloroplastos/química , Codón , Cristalización , Cisteína/química , Datos de Secuencia Molecular , Ácidos Nucleicos/química , Mutación Puntual , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/genética , Homología de Secuencia de Aminoácido , Serina/química , Zea mays/genéticaRESUMEN
SATB1 is essential for T-cell development and growth and metastasis of multitype tumors and acts as a global chromatin organizer and gene expression regulator. The DNA binding ability of SATB1 plays vital roles in its various biological functions. We report the crystal structure of the N-terminal module of SATB1. Interestingly, this module contains a ubiquitin-like domain (ULD) and a CUT repeat-like (CUTL) domain (ULD-CUTL tandem). Detailed biochemical experiments indicate that the N terminus of SATB1 (residues 1-248, SATB1((1-248))), including the extreme 70 N-terminal amino acids, and the ULD-CUTL tandem bind specifically to DNA targets. Our results show that the DNA binding ability of full-length SATB1 requires the contribution of the CUTL domain, as well as the CUT1-CUT2 tandem domain and the homeodomain. These findings may reveal a multiple-domain-coordinated mechanism whereby SATB1 recognizes DNA targets.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión a la Región de Fijación a la Matriz/química , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Secuencia Rica en At , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , UbiquitinaRESUMEN
The highly conserved Paf1 complex (PAF1C) plays critical roles in RNA polymerase II transcription elongation and in the regulation of histone modifications. It has also been implicated in other diverse cellular activities, including posttranscriptional events, embryonic development and cell survival and maintenance of embryonic stem cell identity. Here, we report the structure of the human Paf1/Leo1 subcomplex within PAF1C. The overall structure reveals that the Paf1 and Leo1 subunits form a tightly associated heterodimer through antiparallel beta-sheet interactions. Detailed biochemical experiments indicate that Leo1 binds to PAF1C through Paf1 and that the Ctr9 subunit is the key scaffold protein in assembling PAF1C. Furthermore, we show that the Paf1/Leo1 heterodimer is necessary for its binding to histone H3, the histone octamer, and nucleosome in vitro. Our results shed light on the PAF1C assembly process and substrate recognition during various PAF1C-coordinated histone modifications.
Asunto(s)
Histonas/metabolismo , Proteínas Nucleares/química , Factores de Transcripción/química , Dimerización , Células HEK293 , Humanos , Modelos Moleculares , Proteínas Nucleares/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Special AT-rich sequence-binding protein 1 (SATB1) is a global chromatin organizer and gene expression regulator essential for T-cell development and breast cancer tumor growth and metastasis. The oligomerization of the N-terminal domain of SATB1 is critical for its biological function. We determined the crystal structure of the N-terminal domain of SATB1. Surprisingly, this domain resembles a ubiquitin domain instead of the previously proposed PDZ domain. Our results also reveal that SATB1 can form a tetramer through its N-terminal domain. The tetramerization of SATB1 plays an essential role in its binding to highly specialized DNA sequences. Furthermore, isothermal titration calorimetry results indicate that the SATB1 tetramer can bind simultaneously to two DNA targets. Based on these results, we propose a molecular model whereby SATB1 regulates the expression of multiple genes both locally and at a distance.
Asunto(s)
Proteínas de Unión a la Región de Fijación a la Matriz/química , ADN/metabolismo , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Modelos Moleculares , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de ProteínaRESUMEN
Elongator is a multiprotein complex composed of two subcomplexes, Elp1-3 and Elp4-6. Elongator is highly conserved between yeast and humans and plays an important role in RNA polymerase II-mediated transcriptional elongation and many other processes, including cytoskeleton organization, exocytosis, and tRNA modification. Here, we determined the crystal structure of the Elp4-6 subcomplex of yeast. The overall structure of Elp4-6 revealed that Elp6 acts as a bridge to assemble Elp4 and Elp5. Detailed structural and sequence analyses revealed that each subunit in the Elp4-6 subcomplex forms a RecA-ATPase-like fold, although it lacks the key sequence signature of ATPases. Site-directed mutagenesis and biochemical analyses indicated that the Elp4-6 subcomplex can assemble into a hexameric ring-shaped structure in vitro and in vivo. Furthermore, GST pulldown assays showed that the ring-shaped assembly of the Elp4-6 subcomplex is important for its specific histone H3 binding. Our results may shed light on the substrate recognition and assembly of the holo-Elongator complex.
Asunto(s)
Complejos Multiproteicos/química , Proteínas de Unión al ARN/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Cristalografía por Rayos X , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutagénesis Sitio-Dirigida , Estructura Cuaternaria de Proteína , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The assembly of supramolecular complexes in multidomain scaffold proteins is crucial for the control of cell polarity. The scaffold protein of protein associated with Lin-7 1 (Pals1) forms a complex with two other scaffold proteins, Pals-associated tight junction protein (Patj) and mammalian homolog-2 of Lin-7 (Mals2), through its tandem Lin-2 and Lin-7 (L27) domains to regulate apical-basal polarity. Here, we report the crystal structure of a 4-L27 domain-containing heterotrimer derived from the tripartite complex Patj/Pals1/Mals2. The heterotrimer consists of two cognate pairs of heterodimeric L27 domains with similar conformations. Structural analysis and biochemical data further show that the dimers assemble mutually independently. Additionally, such mutually independent assembly of the two heterodimers can be observed in another tripartite complex, Disks large homolog 1 (DLG1)/calcium-calmodulin-dependent serine protein kinase (CASK)/Mals2. Our results reveal a novel mechanism for tandem L27 domain-mediated, supramolecular complex assembly with a mutually independent mode.
Asunto(s)
Proteínas Portadoras/química , Polaridad Celular , Proteínas de la Membrana/química , Nucleósido-Fosfato Quinasa/química , Multimerización de Proteína , Animales , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Proteínas de la Membrana/metabolismo , Ratones , Modelos Moleculares , Nucleósido-Fosfato Quinasa/metabolismo , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
Pharmacological targeting of endoplasmic reticulum (ER) stress represents one of important methods for disease therapy, which, however, is significantly suppressed by the ER homeostatic processe. Herein, a proof-of-concept strategy is reported for persistent stimulation of ER stress via preventing ER stress adaptation by utilizing multifunctional peptide assemblies. The strategy is established via creation of peptide assemblies with ER-targeting and chaperone glucose-regulated protein 78 (GRP78)-inhibiting functions. The peptides assemblies form well-defined nanofibers that are retrieved by ER organelles in human cervical cancer cell. The underlying mechanism studies unravel that the ER-accumulated peptide assemblies simultaneously stimulate ER stress and inhibit GRP78 refolding activity and thereby promoting endogenous protein aggregation. Combining the internalized peptide assemblies with the induced protein aggregates leads to the persistent stimulation of ER stress. The persistent ER stress induced by the peptide assemblies bestows their application in sensitizing cancer chemotherapy. Both in vitro and in vivo results confirm the enhanced cytotoxicity of drug toyocamycin against HeLa cells by peptide assemblies, thus efficiently inhibiting in vivo tumor growth. The strategy reported here discloses the fundamental keys for efficient promotion of ER stress, thus providing the guidance for development of ER-targeting-assisted cancer chemotherapy in the future.
Asunto(s)
Chaperón BiP del Retículo Endoplásmico , Neoplasias , Humanos , Células HeLa , Estrés del Retículo Endoplásmico , Chaperonas Moleculares , Péptidos/farmacología , Apoptosis , Neoplasias/tratamiento farmacológicoRESUMEN
Kidney development and physiology require polarization of epithelia that line renal tubules. Genetic studies show that polarization of invertebrate epithelia requires the crumbs, partition-defective-3, and discs large complexes. These evolutionarily conserved protein complexes occur in mammalian kidney; however, their role in renal development remains poorly defined. Here, we find that mice lacking the small PDZ protein mammalian LIN-7c (MALS-3) have hypomorphic, cystic, and fibrotic kidneys. Proteomic analysis defines MALS-3 as the only known core component of both the crumbs and discs large cell polarity complexes. MALS-3 mediates stable assembly of the crumbs tight junction complex and the discs large basolateral complex, and these complexes are disrupted in renal epithelia from MALS-3 knockout mice. Interestingly, MALS-3 controls apico-basal polarity preferentially in epithelia derived from metanephric mesenchyme, and defects in kidney architecture owe solely to MALS expression in these epithelia. These studies demonstrate that defects in epithelial cell polarization can cause cystic and fibrotic renal disease.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Riñón/patología , Complejos Multiproteicos/fisiología , Proteínas Adaptadoras Transductoras de Señales/fisiología , Secuencia de Aminoácidos , Animales , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/fisiología , Proteínas de Ciclo Celular , Células Epiteliales/metabolismo , Riñón/embriología , Riñón/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Organogénesis/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Alineación de Secuencia , Uniones Estrechas/metabolismo , Uniones Estrechas/patologíaRESUMEN
The highly conserved multifunctional polymerase-associated factor 1 (Paf1) complex (PAF1C), which consists of five core subunits: Ctr9, Paf1, Leo1, Cdc73, and Rtf1, acts as a diverse hub that regulates all stages of RNA polymerase II-mediated transcription and various other cellular functions. However, the underlying mechanisms remain unclear. Here, we report the crystal structure of the core module derived from a quaternary Ctr9/Paf1/Cdc73/Rtf1 complex of S. cerevisiae PAF1C, which reveals interfaces between the tetratricopeptide repeat module in Ctr9 and Cdc73 or Rtf1, and find that the Ctr9/Paf1 subcomplex is the key scaffold for PAF1C assembly. Our study demonstrates that Cdc73 binds Ctr9/Paf1 subcomplex with a very similar conformation within thermophilic fungi or human PAF1C, and that the binding of Cdc73 to PAF1C is important for yeast growth. Importantly, our structure reveals for the first time that the extreme C-terminus of Rtf1 adopts an "L"-shaped structure, which interacts with Ctr9 specifically. In addition, disruption of the binding of either Cdc73 or Rtf1 to PAF1C greatly affects the normal level of histone H2B K123 monoubiquitination in vivo. Collectively, our results provide a structural insight into the architecture of the quaternary Ctr9/Paf1/Cdc73/Rtf1 complex and PAF1C functional regulation.