RESUMO
Opioids such as morphine and oxycodone are analgesics frequently prescribed for the treatment of moderate or severe pain. Unfortunately, these medications are associated with exceptionally high abuse potentials and often cause fatal side effects, mainly through the µ-opioid receptor (MOR). Efforts to discover novel, safer, and more efficacious analgesics targeting MOR have encountered challenges. In this review, we summarize alternative strategies and targets that could be used to develop safer nonopioid analgesics. A molecular understanding of G protein-coupled receptor activation and signaling has illuminated not only the complexities of receptor pharmacology but also the potential for pathway-selective agonists and allosteric modulators as safer medications. The availability of structures of pain-related receptors, in combination with high-throughput computational tools, has accelerated the discovery of multitarget ligands with promising pharmacological profiles. Emerging clinical evidence also supports the notion that drugs targeting peripheral opioid receptors have potential as improved analgesic agents.
Assuntos
Analgésicos não Narcóticos/química , Analgésicos não Narcóticos/farmacologia , Receptores Opioides/química , Receptores Opioides/metabolismo , Analgésicos Opioides/efeitos adversos , Analgésicos Opioides/farmacologia , Animais , Descoberta de Drogas , Ensaios de Triagem em Larga Escala/métodos , Humanos , Ligantes , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.
Assuntos
Elementos Facilitadores Genéticos/genética , Ensaios de Triagem em Larga Escala/métodos , Polidactilia/genética , Animais , Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Introdução de Genes/métodos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Mutação , Fenótipo , Polidactilia/metabolismo , RNA não Traduzido/genéticaRESUMO
The 3D organization of chromatin regulates many genome functions. Our understanding of 3D genome organization requires tools to directly visualize chromatin conformation in its native context. Here we report an imaging technology for visualizing chromatin organization across multiple scales in single cells with high genomic throughput. First we demonstrate multiplexed imaging of hundreds of genomic loci by sequential hybridization, which allows high-resolution conformation tracing of whole chromosomes. Next we report a multiplexed error-robust fluorescence in situ hybridization (MERFISH)-based method for genome-scale chromatin tracing and demonstrate simultaneous imaging of more than 1,000 genomic loci and nascent transcripts of more than 1,000 genes together with landmark nuclear structures. Using this technology, we characterize chromatin domains, compartments, and trans-chromosomal interactions and their relationship to transcription in single cells. We envision broad application of this high-throughput, multi-scale, and multi-modal imaging technology, which provides an integrated view of chromatin organization in its native structural and functional context.
Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , Cromossomos Humanos/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Hibridização in Situ Fluorescente/métodos , Análise de Célula Única/métodos , Algoritmos , Linhagem Celular , Núcleo Celular/genética , Cromatina/genética , Cromossomos Humanos/genética , DNA/genética , DNA/metabolismo , Genômica , Humanos , Processamento de Imagem Assistida por Computador , Conformação Molecular , Imagem Multimodal , Região Organizadora do Nucléolo/genética , Região Organizadora do Nucléolo/metabolismo , RNA/genética , RNA/metabolismo , SoftwareRESUMO
The precise control of CRISPR-Cas9 activity is required for a number of genome engineering technologies. Here, we report a generalizable platform that provided the first synthetic small-molecule inhibitors of Streptococcus pyogenes Cas9 (SpCas9) that weigh <500 Da and are cell permeable, reversible, and stable under physiological conditions. We developed a suite of high-throughput assays for SpCas9 functions, including a primary screening assay for SpCas9 binding to the protospacer adjacent motif, and used these assays to screen a structurally diverse collection of natural-product-like small molecules to ultimately identify compounds that disrupt the SpCas9-DNA interaction. Using these synthetic anti-CRISPR small molecules, we demonstrated dose and temporal control of SpCas9 and catalytically impaired SpCas9 technologies, including transcription activation, and identified a pharmacophore for SpCas9 inhibition using structure-activity relationships. These studies establish a platform for rapidly identifying synthetic, miniature, cell-permeable, and reversible inhibitors against both SpCas9 and next-generation CRISPR-associated nucleases.
Assuntos
Proteína 9 Associada à CRISPR/antagonistas & inibidores , Sistemas CRISPR-Cas/fisiologia , Ensaios de Triagem em Larga Escala/métodos , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , DNA/metabolismo , Endonucleases/metabolismo , Edição de Genes/métodos , Genoma , Bibliotecas de Moléculas Pequenas , Streptococcus pyogenes/genética , Especificidade por SubstratoRESUMO
B cell receptor (BCR) sequencing is a powerful tool for interrogating immune responses to infection and vaccination, but it provides limited information about the antigen specificity of the sequenced BCRs. Here, we present LIBRA-seq (linking B cell receptor to antigen specificity through sequencing), a technology for high-throughput mapping of paired heavy- and light-chain BCR sequences to their cognate antigen specificities. B cells are mixed with a panel of DNA-barcoded antigens so that both the antigen barcode(s) and BCR sequence are recovered via single-cell next-generation sequencing. Using LIBRA-seq, we mapped the antigen specificity of thousands of B cells from two HIV-infected subjects. The predicted specificities were confirmed for a number of HIV- and influenza-specific antibodies, including known and novel broadly neutralizing antibodies. LIBRA-seq will be an integral tool for antibody discovery and vaccine development efforts against a wide range of antigen targets.
Assuntos
Mapeamento de Epitopos/métodos , Epitopos/química , Receptores de Antígenos de Linfócitos B/química , Análise de Sequência de DNA/métodos , Análise de Célula Única/métodos , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Antígenos/química , Antígenos/imunologia , Células Cultivadas , Epitopos/imunologia , Células HEK293 , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Receptores de Antígenos de Linfócitos B/imunologia , Células THP-1RESUMO
A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell sorting at an unprecedented rate. This technology, which we refer to as intelligent image-activated cell sorting, integrates high-throughput cell microscopy, focusing, and sorting on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. We use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.
Assuntos
Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala/métodos , Processamento de Imagem Assistida por Computador/métodos , Animais , Aprendizado Profundo , HumanosRESUMO
Peptides have great potential to combat antibiotic resistance. While many platforms can screen peptides for their ability to bind to target cells, there are virtually no platforms that directly assess the functionality of peptides. This limitation is exacerbated when identifying antimicrobial peptides because the phenotype, death, selects against itself and has caused a scientific bottleneck that confines research to a few naturally occurring classes of antimicrobial peptides. We have used this seeming dissonance to develop Surface Localized Antimicrobial Display (SLAY), a platform that allows screening of unlimited numbers of peptides of any length, composition, and structure in a single tube for antimicrobial activity. Using SLAY, we screened â¼800,000 random peptide sequences for antimicrobial function and identified thousands of active sequences, dramatically increasing the number of known antimicrobial sequences. SLAY hits present with different potential mechanisms of peptide action and access to areas of antimicrobial physicochemical space beyond what nature has evolved. VIDEO ABSTRACT.
Assuntos
Antibacterianos/farmacologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Biblioteca de Peptídeos , Animais , Antibacterianos/química , Escherichia coli , CamundongosRESUMO
By developing a high-density murine immunophenotyping platform compatible with high-throughput genetic screening, we have established profound contributions of genetics and structure to immune variation (http://www.immunophenotype.org). Specifically, high-throughput phenotyping of 530 unique mouse gene knockouts identified 140 monogenic 'hits', of which most had no previous immunologic association. Furthermore, hits were collectively enriched in genes for which humans show poor tolerance to loss of function. The immunophenotyping platform also exposed dense correlation networks linking immune parameters with each other and with specific physiologic traits. Such linkages limit freedom of movement for individual immune parameters, thereby imposing genetically regulated 'immunologic structures', the integrity of which was associated with immunocompetence. Hence, we provide an expanded genetic resource and structural perspective for understanding and monitoring immune variation in health and disease.
Assuntos
Infecções por Enterobacteriaceae/imunologia , Variação Genética/genética , Ensaios de Triagem em Larga Escala/métodos , Imunofenotipagem/métodos , Infecções por Salmonella/imunologia , Animais , Citrobacter/imunologia , Infecções por Enterobacteriaceae/microbiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Salmonella/imunologia , Infecções por Salmonella/microbiologiaRESUMO
The construction of synthetic gene circuits requires the rational combination of multiple regulatory components, but predicting their behavior can be challenging due to poorly understood component interactions and unexpected emergent behaviors. In eukaryotes, chromatin regulators (CRs) are essential regulatory components that orchestrate gene expression. Here, we develop a screening platform to investigate the impact of CR pairs on transcriptional activity in yeast. We construct a combinatorial library consisting of over 1,900 CR pairs and use a high-throughput workflow to characterize the impact of CR co-recruitment on gene expression. We recapitulate known interactions and discover several instances of CR pairs with emergent behaviors. We also demonstrate that supervised machine learning models trained with low-dimensional amino acid embeddings accurately predict the impact of CR co-recruitment on transcriptional activity. This work introduces a scalable platform and machine learning approach that can be used to study how networks of regulatory components impact gene expression.
Assuntos
Cromatina , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Saccharomyces cerevisiae , Biologia Sintética , Transcrição Gênica , Cromatina/metabolismo , Cromatina/genética , Biologia Sintética/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Aprendizado de Máquina Supervisionado , Montagem e Desmontagem da Cromatina , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
The human genome functions as a three-dimensional chromatin polymer, driven by a complex collection of chromosome interactions1-3. Although the molecular rules governing these interactions are being quickly elucidated, relatively few proteins regulating this process have been identified. Here, to address this gap, we developed high-throughput DNA or RNA labelling with optimized Oligopaints (HiDRO)-an automated imaging pipeline that enables the quantitative measurement of chromatin interactions in single cells across thousands of samples. By screening the human druggable genome, we identified more than 300 factors that influence genome folding during interphase. Among these, 43 genes were validated as either increasing or decreasing interactions between topologically associating domains. Our findings show that genetic or chemical inhibition of the ubiquitous kinase GSK3A leads to increased long-range chromatin looping interactions in a genome-wide and cohesin-dependent manner. These results demonstrate the importance of GSK3A signalling in nuclear architecture and the use of HiDRO for identifying mechanisms of spatial genome organization.
Assuntos
Cromatina , Posicionamento Cromossômico , Cromossomos Humanos , Genoma Humano , Quinases da Glicogênio Sintase , Ensaios de Triagem em Larga Escala , Análise de Célula Única , Humanos , Cromatina/efeitos dos fármacos , Cromatina/genética , Cromatina/metabolismo , Posicionamento Cromossômico/efeitos dos fármacos , Cromossomos Humanos/efeitos dos fármacos , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , DNA/análise , DNA/metabolismo , Genoma Humano/efeitos dos fármacos , Genoma Humano/genética , Quinases da Glicogênio Sintase/antagonistas & inibidores , Quinases da Glicogênio Sintase/deficiência , Quinases da Glicogênio Sintase/genética , Ensaios de Triagem em Larga Escala/métodos , Interfase , Reprodutibilidade dos Testes , RNA/análise , RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única/métodos , CoesinasRESUMO
Acidic transcription activation domains (ADs) are encoded by a wide range of seemingly unrelated amino acid sequences, making it difficult to recognize features that promote their dynamic behavior, "fuzzy" interactions, and target specificity. We screened a large set of random 30-mer peptides for AD function in yeast and trained a deep neural network (ADpred) on the AD-positive and -negative sequences. ADpred identifies known acidic ADs within transcription factors and accurately predicts the consequences of mutations. Our work reveals that strong acidic ADs contain multiple clusters of hydrophobic residues near acidic side chains, explaining why ADs often have a biased amino acid composition. ADs likely use a binding mechanism similar to avidity where a minimum number of weak dynamic interactions are required between activator and target to generate biologically relevant affinity and in vivo function. This mechanism explains the basis for fuzzy binding observed between acidic ADs and targets.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Fatores de Transcrição/genética , Ativação Transcricional/genética , Sequência de Aminoácidos/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas de Ligação a DNA/metabolismo , Aprendizado Profundo , Ligação Proteica , Domínios Proteicos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/fisiologiaRESUMO
Traditionally, chemical toxicity is determined by in vivo animal studies, which are low throughput, expensive, and sometimes fail to predict compound toxicity in humans. Due to the increasing number of chemicals in use and the high rate of drug candidate failure due to toxicity, it is imperative to develop in vitro, high-throughput screening methods to determine toxicity. The Tox21 program, a unique research consortium of federal public health agencies, was established to address and identify toxicity concerns in a high-throughput, concentration-responsive manner using a battery of in vitro assays. In this article, we review the advancements in high-throughput robotic screening methodology and informatics processes to enable the generation of toxicological data, and their impact on the field; further, we discuss the future of assessing environmental toxicity utilizing efficient and scalable methods that better represent the corresponding biological and toxicodynamic processes in humans.
Assuntos
Ensaios de Triagem em Larga Escala , Toxicologia , Animais , Humanos , Ensaios de Triagem em Larga Escala/métodos , Toxicologia/métodosRESUMO
Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here we describe the high-throughput, functional assessment of phosphorylation sites through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of PHLPP1, which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.
Assuntos
Processamento de Proteína Pós-Traducional , Fosforilação , Humanos , Fatores de Transcrição NFATC/metabolismo , Fatores de Transcrição NFATC/genética , Transdução de Sinais , Células HEK293 , Proteômica/métodos , Ensaios de Triagem em Larga Escala/métodos , Linfócitos T/metabolismo , Células Jurkat , NF-kappa B/metabolismoRESUMO
Throughout history, humans have relied on plants as a source of medication, flavoring, and food. Plants synthesize large chemical libraries and release many of these compounds into the rhizosphere and atmosphere where they affect animal and microbe behavior. To survive, nematodes must have evolved the sensory capacity to distinguish plant-made small molecules (SMs) that are harmful and must be avoided from those that are beneficial and should be sought. This ability to classify chemical cues as a function of their value is fundamental to olfaction and represents a capacity shared by many animals, including humans. Here, we present an efficient platform based on multiwell plates, liquid handling instrumentation, inexpensive optical scanners, and bespoke software that can efficiently determine the valence (attraction or repulsion) of single SMs in the model nematode, Caenorhabditis elegans. Using this integrated hardware-wetware-software platform, we screened 90 plant SMs and identified 37 that attracted or repelled wild-type animals but had no effect on mutants defective in chemosensory transduction. Genetic dissection indicates that for at least 10 of these SMs, response valence emerges from the integration of opposing signals, arguing that olfactory valence is often determined by integrating chemosensory signals over multiple lines of information. This study establishes that C. elegans is an effective discovery engine for determining chemotaxis valence and for identifying natural products detected by the chemosensory nervous system.
Assuntos
Caenorhabditis elegans , Quimiotaxia , Ensaios de Triagem em Larga Escala , Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/efeitos dos fármacos , Animais , Ensaios de Triagem em Larga Escala/métodos , Olfato/fisiologia , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , SoftwareRESUMO
Trained immunity is characterized by epigenetic and metabolic reprogramming in response to specific stimuli. This rewiring can result in increased cytokine and effector responses to pathogenic challenges, providing nonspecific protection against disease. It may also improve immune responses to established immunotherapeutics and vaccines. Despite its promise for next-generation therapeutic design, most current understanding and experimentation is conducted with complex and heterogeneous biologically derived molecules, such as ß-glucan or the Bacillus Calmette-Guérin (BCG) vaccine. This limited collection of training compounds also limits the study of the genes most involved in training responses as each molecule has both training and nontraining effects. Small molecules with tunable pharmacokinetics and delivery modalities would both assist in the study of trained immunity and its future applications. To identify small molecule inducers of trained immunity, we screened a library of 2,000 drugs and drug-like compounds. Identification of well-defined compounds can improve our understanding of innate immune memory and broaden the scope of its clinical applications. We identified over two dozen small molecules in several chemical classes that induce a training phenotype in the absence of initial immune activation-a current limitation of reported inducers of training. A surprising result was the identification of glucocorticoids, traditionally considered immunosuppressive, providing an unprecedented link between glucocorticoids and trained innate immunity. We chose seven of these top candidates to characterize and establish training activity in vivo. In this work, we expand the number of compounds known to induce trained immunity, creating alternative avenues for studying and applying innate immune training.
Assuntos
Ensaios de Triagem em Larga Escala , Imunidade Inata , Bibliotecas de Moléculas Pequenas , Animais , Camundongos , Ensaios de Triagem em Larga Escala/métodos , Imunidade Inata/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Camundongos Endogâmicos C57BL , Memória Imunológica/efeitos dos fármacos , Imunidade TreinadaRESUMO
Seasonal and pandemic-associated influenza strains cause highly contagious viral respiratory infections that can lead to severe illness and excess mortality. Here, we report on the optimization of our small-molecule inhibitor F0045(S) targeting the influenza hemagglutinin (HA) stem with our Sulfur-Fluoride Exchange (SuFEx) click chemistry-based high-throughput medicinal chemistry (HTMC) strategy. A combination of SuFEx- and amide-based lead molecule diversification and structure-guided design led to identification and validation of ultrapotent influenza fusion inhibitors with subnanomolar EC50 cellular antiviral activity against several influenza A group 1 strains. X-ray structures of six of these compounds with HA indicate that the appended moieties occupy additional pockets on the HA surface and increase the binding interaction, where the accumulation of several polar interactions also contributes to the improved affinity. The compounds here represent the most potent HA small-molecule inhibitors to date. Our divergent HTMC platform is therefore a powerful, rapid, and cost-effective approach to develop bioactive chemical probes and drug-like candidates against viral targets.
Assuntos
Antivirais , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Antivirais/farmacologia , Antivirais/química , Química Farmacêutica/métodos , Ensaios de Triagem em Larga Escala/métodos , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Cristalografia por Raios X/métodos , Química Click/métodos , Animais , Vírus da Influenza A/efeitos dos fármacos , Células Madin Darby de Rim Canino , Inibidores de Proteínas Virais de Fusão/farmacologia , Inibidores de Proteínas Virais de Fusão/química , CãesRESUMO
The caspases are unique proteases that mediate the major morphological changes of apoptosis and various other cellular remodeling processes. As we catalog and study the myriad proteins subject to cleavage by caspases, we are beginning to appreciate the full functional repertoire of these enzymes. Here, we examine current knowledge about caspase cleavages: what kinds of proteins are cut, in what contexts, and to what end. After reviewing basic caspase biology, we describe the technologies that enable high-throughput caspase substrate discovery and the datasets they have yielded. We discuss how caspases recognize their substrates and how cleavages are conserved among different metazoan organisms. Rather than comprehensively reviewing all known substrates, we use examples to highlight some functional impacts of caspase cuts during apoptosis and differentiation. Finally, we discuss the roles caspase substrates can play in medicine. Though great progress has been made in this field, many important areas still await exploration.
Assuntos
Apoptose/fisiologia , Caspases/química , Caspases/metabolismo , Diferenciação Celular/fisiologia , Animais , Caspases/classificação , Caspases/genética , Dimerização , Ensaios de Triagem em Larga Escala/métodos , Humanos , Modelos Moleculares , Conformação Proteica , Transdução de Sinais/fisiologia , Especificidade por SubstratoRESUMO
Nucleosomes present a barrier for the binding of most transcription factors (TFs). However, special TFs known as nucleosome-displacing factors (NDFs) can access embedded sites and cause the depletion of the local nucleosomes as well as repositioning of the neighboring nucleosomes. Here, we developed a novel high-throughput method in yeast to identify NDFs among 104 TFs and systematically characterized the impact of orientation, affinity, location, and copy number of their binding motifs on the nucleosome occupancy. Using this assay, we identified 29 NDF motifs and divided the nuclear TFs into three groups with strong, weak, and no nucleosome-displacing activities. Further studies revealed that tight DNA binding is the key property that underlies NDF activity, and the NDFs may partially rely on the DNA replication to compete with nucleosome. Overall, our study presents a framework to functionally characterize NDFs and elucidate the mechanism of nucleosome invasion.
Assuntos
Nucleossomos/metabolismo , Saccharomycetales/metabolismo , Cromatina/metabolismo , Replicação do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Histonas/metabolismo , Humanos , Modelos Moleculares , Nucleossomos/genética , Regiões Promotoras Genéticas , Ligação Proteica , Multimerização Proteica , Saccharomycetales/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
We have developed a highly parallel strategy, systematic gene-to-phenotype arrays (SGPAs), to comprehensively map the genetic landscape driving molecular phenotypes of interest. By this approach, a complete yeast genetic mutant array is crossed with fluorescent reporters and imaged on membranes at high density and contrast. Importantly, SGPA enables quantification of phenotypes that are not readily detectable in ordinary genetic analysis of cell fitness. We benchmark SGPA by examining two fundamental biological phenotypes: first, we explore glucose repression, in which SGPA identifies a requirement for the Mediator complex and a role for the CDK8/kinase module in regulating transcription. Second, we examine selective protein quality control, in which SGPA identifies most known quality control factors along with U34 tRNA modification, which acts independently of proteasomal degradation to limit misfolded protein production. Integration of SGPA with other fluorescent readouts will enable genetic dissection of a wide range of biological pathways and conditions.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/métodos , Quinase 8 Dependente de Ciclina/genética , Redes Reguladoras de Genes , Genótipo , Complexo Mediador/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Protein identification and quantification is an important tool for biomarker discovery. With the increased sensitivity and speed of modern mass spectrometers, sample preparation remains a bottleneck for studying large cohorts. To address this issue, we prepared and evaluated a simple and efficient workflow on the Opentrons OT-2 robot that combines sample digestion, cleanup, and loading on Evotips in a fully automated manner, allowing the processing of up to 192 samples in 6 h. Analysis of 192 automated HeLa cell sample preparations consistently identified â¼8000 protein groups and â¼130,000 peptide precursors with an 11.5 min active liquid chromatography gradient with the Evosep One and narrow-window data-independent acquisition (nDIA) with the Orbitrap Astral mass spectrometer providing a throughput of 100 samples per day. Our results demonstrate a highly sensitive workflow yielding both reproducibility and stability at low sample inputs. The workflow is optimized for minimal sample starting amount to reduce the costs for reagents needed for sample preparation, which is critical when analyzing large biological cohorts. Building on the digesting workflow, we incorporated an automated phosphopeptide enrichment step using magnetic titanium-immobilized metal ion affinity chromatography beads. This allows for a fully automated proteome and phosphoproteome sample preparation in a single step with high sensitivity. Using the integrated digestion and Evotip loading workflow, we evaluated the effects of cancer immune therapy on the plasma proteome in metastatic melanoma patients.