RESUMEN
Polycomb repressive complex 2 (PRC2) is reported to bind to many RNAs and has become a central player in reports of how long non-coding RNAs (lncRNAs) regulate gene expression. Yet, there is a growing discrepancy between the biochemical evidence supporting specific lncRNA-PRC2 interactions and functional evidence demonstrating that PRC2 is often dispensable for lncRNA function. Here, we revisit the evidence supporting RNA binding by PRC2 and show that many reported interactions may not occur in vivo. Using denaturing purification of in vivo crosslinked RNA-protein complexes in human and mouse cell lines, we observe a loss of detectable RNA binding to PRC2 and chromatin-associated proteins previously reported to bind RNA (CTCF, YY1, and others), despite accurately mapping bona fide RNA-binding sites across others (SPEN, TET2, and others). Taken together, these results argue for a critical re-evaluation of the broad role of RNA binding to orchestrate various chromatin regulatory mechanisms.
Asunto(s)
Complejo Represivo Polycomb 2 , ARN Largo no Codificante , Animales , Ratones , Humanos , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Cromatina/genética , Sitios de UniónRESUMEN
The α-helix is one of the most common protein surface recognition motifs found in nature, and its unique amide-cloaking properties also enable α-helical polypeptide motifs to exist in membranes. Together, these properties have inspired the development of α-helically constrained (Helicon) therapeutics that can enter cells and bind targets that have been considered "undruggable", such as protein-protein interactions. To date, no general method for discovering α-helical binders to proteins has been reported, limiting Helicon drug discovery to only those proteins with previously characterized α-helix recognition sites, and restricting the starting chemical matter to those known α-helical binders. Here, we report a general and rapid screening method to empirically map the α-helix binding sites on a broad range of target proteins in parallel using large, unbiased Helicon phage display libraries and next-generation sequencing. We apply this method to screen six structurally diverse protein domains, only one of which had been previously reported to bind isolated α-helical peptides, discovering 20 families that collectively comprise several hundred individual Helicons. Analysis of 14 X-ray cocrystal structures reveals at least nine distinct α-helix recognition sites across these six proteins, and biochemical and biophysical studies show that these Helicons can block protein-protein interactions, inhibit enzymatic activity, induce conformational rearrangements, and cause protein dimerization. We anticipate that this method will prove broadly useful for the study of protein recognition and for the development of both biochemical tools and therapeutics for traditionally challenging protein targets.
Asunto(s)
Amidas , Péptidos , Conformación Proteica en Hélice alfa , Sitios de Unión , Péptidos/química , Biblioteca de PéptidosRESUMEN
SynGAP is a Ras and Rap GTPase-activating protein (GAP) found in high concentration in the postsynaptic density (PSD) fraction from mammalian forebrain where it binds to PDZ domains of PSD-95. Phosphorylation of pure recombinant synGAP by Ca2+/calmodulin-dependent protein kinase II (CaMKII) shifts the balance of synGAP's GAP activity toward inactivation of Rap1; whereas phosphorylation by cyclin-dependent kinase 5 (CDK5) has the opposite effect, shifting the balance toward inactivation of HRas. These shifts in balance contribute to regulation of the numbers of surface AMPA receptors, which rise during synaptic potentiation (CaMKII) and fall during synaptic scaling (CDK5). Polo-like kinase 2 (Plk2/SNK), like CDK5, contributes to synaptic scaling. These two kinases act in concert to reduce the number of surface AMPA receptors following elevated neuronal activity by tagging spine-associated RapGAP protein (SPAR) for degradation, thus raising the level of activated Rap. Here we show that Plk2 also phosphorylates and regulates synGAP. Phosphorylation of synGAP by Plk2 stimulates its GAP activity toward HRas by 65%, and toward Rap1 by 16%. Simultaneous phosphorylation of synGAP by Plk2 and CDK5 at distinct sites produces an additive increase in GAP activity toward HRas (â¼230%) and a smaller, non-additive increase in activity toward Rap1 (â¼15%). Dual phosphorylation also produces an increase in GAP activity toward Rap2 (â¼40-50%), an effect not produced by either kinase alone. As we previously observed for CDK5, addition of Ca2+/CaM causes a substrate-directed doubling of the rate and stoichiometry of phosphorylation of synGAP by Plk2, targeting residues also phosphorylated by CaMKII. In summary, phosphorylation by Plk2, like CDK5, shifts the ratio of GAP activity of synGAP to produce a greater decrease in active Ras than in active Rap, which would produce a shift toward a decrease in the number of surface AMPA receptors in neuronal dendrites.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Animales , Células COS , Chlorocebus aethiops , Humanos , Espectrometría de Masas , FosforilaciónRESUMEN
synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Quinasa 5 Dependiente de la Ciclina , Proteínas Activadoras de GTPasa , Proteínas Oncogénicas , Proteínas Proto-Oncogénicas p21(ras) , Sinapsis/enzimología , Proteínas de Unión al GTP rap , Proteínas de Unión al GTP rap1 , Proteínas Activadoras de ras GTPasa , Proteínas ras , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Células Cultivadas , Quinasa 5 Dependiente de la Ciclina/química , Quinasa 5 Dependiente de la Ciclina/genética , Quinasa 5 Dependiente de la Ciclina/metabolismo , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Neuronas/citología , Neuronas/enzimología , Proteínas Oncogénicas/química , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ratas , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas de Unión al GTP rap1/química , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismo , Proteínas Activadoras de ras GTPasa/química , Proteínas Activadoras de ras GTPasa/genética , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise â¼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and ß-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins.
Asunto(s)
Cromatografía de Afinidad/métodos , Proteínas/química , Proteínas/aislamiento & purificación , Cromatografía de Afinidad/instrumentación , Humanos , Ligandos , Dominios PDZ , Ingeniería de Proteínas , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Adaptive changes in lysosomal capacity are driven by the transcription factors TFEB and TFE3 in response to increased autophagic flux and endolysosomal stress, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolysosomal system. Here, we show that during the conjugation of ATG8 to single membranes (CASM), Parkin-dependent mitophagy, and Salmonella-induced xenophagy, the membrane conjugation of GABARAP, but not LC3, is required for activation of TFEB/TFE3 to control lysosomal capacity. GABARAP directly binds to a previously unidentified LC3-interacting motif (LIR) in the FLCN/FNIP tumor suppressor complex and mediates sequestration to GABARAP-conjugated membrane compartments. This disrupts FLCN/FNIP GAP function toward RagC/D, resulting in impaired substrate-specific mTOR-dependent phosphorylation of TFEB. Thus, the GABARAP-FLCN/FNIP-TFEB axis serves as a molecular sensor that coordinates lysosomal homeostasis with perturbations and cargo flux within the autophagy-lysosomal network.
RESUMEN
Criteria for sub-typing of microbial organisms by DNA sequencing proposed by Olive and Bean were applied to several genes in Escherichia coli to identify targets for the development of microbial source tracking assays. Based on the aforementioned criteria, the icd (isocitrate dehydrogenase), and putP (proline permease) genes were excluded as potential targets due to their high rates of horizontal gene transfer; the rrs (16S rRNA) gene was excluded as a target due to the presence of multiple gene copies, with different sequences in a single genome. Based on the above criteria, the mdh (malate dehydrogenase) gene was selected as a target for development of a microbial source tracking assay. The mdh assay was optimized to analyze a 150 bp fragment corresponding to residues G191 to R240 (helices H10 and H11) of the Mdh catalytic domain. 295 fecal isolates (52 horse, 50 deer, 72 dog, 52 seagull and 69 human isolates) were sequenced and analyzed. Target DNA sequences for isolates from horse, dog plus deer, and seagull formed identifiable groupings. Sequences from human isolates, aside from a low level (ca. 15%) human specific sequence, did not group; nevertheless, other hosts could be distinguished from human. Positive and negative predictive values for two- and three-way host comparisons ranged from 60% to 90% depending on the focus host. False positive rates were below 10%. Multiple E. coli isolates from individual fecal samples exhibited high levels of sequence homogeneity, i.e. typically only one to two mdh sequences were observed per up to five E. coli isolates from a single fecal sample. Among all isolates sequenced from fecal samples from each host, sequence homogeneity decreased in the following order: horse>dog>deer>human and gull. For in-library isolates, blind analysis of fecal isolates (n=12) from four hosts known to contain host specific target sequences was 100% accurate and 100% reproducible for both DNA sequence and host identification. For blind analysis of non-library isolates, 18/19 isolates (94.7%) matched one or more library sequences for the corresponding host. Ten of eleven geographical outlier fecal isolates from Florida had mdh sequences that were identical to in-library sequences for the corresponding host from California. The mdh assay was successfully applied to environmental isolates from an underground telephone vault in California, with 4 of 5 isolates matching sequences in the mdh library. 146 sequences of the 645bp mdh fragment from five host sources were translated into protein sequence and aligned. Seven unique Mdh protein sequences, which contained eight polymorphic sites, were identified. Six of the polymorphic sites were in the NAD+ binding domain and two were in the catalytic domain. All of the polymorphic sites were located in surface exposed regions of the protein. None of the non-silent mutations of the Mdh protein were in the 150bp mdh target. The advantages and disadvantages of the assay compared to established source tracking methods are discussed.
Asunto(s)
Técnicas de Tipificación Bacteriana , Infecciones por Escherichia coli/microbiología , Escherichia coli/clasificación , Escherichia coli/genética , Malato Deshidrogenasa/genética , Epidemiología Molecular/métodos , Análisis de Secuencia de ADN , Animales , Secuencia de Bases , Dominio Catalítico/genética , Charadriiformes/microbiología , Ciervos/microbiología , Perros , Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Heces/microbiología , Genes Bacterianos , Caballos/microbiología , Humanos , Malato Deshidrogenasa/química , Datos de Secuencia Molecular , Mutación , Polimorfismo Genético , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Homología de Secuencia de Ácido NucleicoRESUMEN
SynGAP is a Ras/Rap GTPase-activating protein (GAP) that is a major constituent of postsynaptic densities (PSDs) from mammalian forebrain. Its α1 isoform binds to all three PDZ (PSD-95, Discs-large, ZO-1) domains of PSD-95, the principal PSD scaffold, and can occupy as many as 15% of these PDZ domains. We present evidence that synGAP-α1 regulates the composition of the PSD by restricting binding to the PDZ domains of PSD-95. We show that phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and Polo-like kinase-2 (PLK2) decreases its affinity for the PDZ domains by several fold, which would free PDZ domains for occupancy by other proteins. Finally, we show that three critical postsynaptic signaling proteins that bind to the PDZ domains of PSD-95 are present in higher concentration in PSDs isolated from mice with a heterozygous deletion of synGAP.
Asunto(s)
Homólogo 4 de la Proteína Discs Large/metabolismo , Dominios PDZ , Densidad Postsináptica/química , Proteínas Activadoras de ras GTPasa/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Ratones , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
The quantification of plasmid DNA by the PicoGreen dye binding assay has been automated, and the effect of quantification of user-submitted templates on DNA sequence quality in a core laboratory has been assessed. The protocol pipets, mixes and reads standards, blanks and up to 88 unknowns, generates a standard curve, and calculates template concentrations. For pUC19 replicates at five concentrations, coefficients of variance were 0.1, and percent errors were from 1% to 7% (n=198). Standard curves with pUC19 DNA were nonlinear over the 1 to 1733 ng/microL concentration range required to assay the majority (98.7%) of user-submitted templates. Over 35,000 templates have been quantified using the protocol. For 1350 user-submitted plasmids, 87% deviated by >or=20% from the requested concentration (500 ng/microL). Based on data from 418 sequencing reactions, quantification of user-submitted templates was shown to significantly improve DNA sequence quality. The protocol is applicable to all types of double-stranded DNA, is unaffected by primer (1 pmol/microL), and is user modifiable. The protocol takes 30 min, saves 1 h of technical time, and costs approximately $0.20 per unknown.
Asunto(s)
Análisis de Secuencia de ADN/métodos , Plásmidos , Moldes GenéticosRESUMEN
PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands.
Asunto(s)
Cromatografía de Afinidad/métodos , Dominios PDZ , Proteínas/aislamiento & purificación , Marcadores de Afinidad , Secuencia de Aminoácidos , Animales , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Proton-conducting materials play a central role in many renewable energy and bioelectronics technologies, including fuel cells, batteries and sensors. Thus, much research effort has been expended to develop improved proton-conducting materials, such as ceramic oxides, solid acids, polymers and metal-organic frameworks. Within this context, bulk proton conductors from naturally occurring proteins have received somewhat less attention than other materials, which is surprising given the potential modularity, tunability and processability of protein-based materials. Here, we report proton conductivity for thin films composed of reflectin, a cephalopod structural protein. Bulk reflectin has a proton conductivity of ~2.6 × 10(-3) S cm(-1) at 65 °C, a proton transport activation energy of ~0.2 eV and a proton mobility of ~7 × 10(-3) cm(2) V(-1) s(-1). These figures of merit are similar to those reported for state-of-the-art artificial proton conductors and make it possible to use reflectin in protein-based protonic transistors. Our findings may hold implications for the next generation of biocompatible proton-conducting materials and protonic devices.
Asunto(s)
Cefalópodos/química , Polímeros/química , Terapia de Protones , AnimalesRESUMEN
In nature, cephalopods employ unique dynamic camouflage mechanisms. Herein, we draw inspiration from self-assembled structures found in cephalopods to fabricate tunable biomimetic camouflage coatings. The reflectance of these coatings is dynamically modulated between the visible and infrared regions of the electromagnetic spectrum in situ. Our studies represent a crucial step towards reconfigurable and disposable infrared camouflage for stealth applications.