RESUMO
To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures in vitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.0-Å reconstructions of axonemal microtubule structures. The well-resolved tertiary structures allowed us to unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105, and SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules, suggesting their roles in modulating the mechanical properties of the filaments. Indeed, Tekt5 -/- sperm possess more deformed flagella with 180° bends. Together, our studies presented a cellular visual proteomics workflow and shed light on the in vivo functions of Tektin 5.
Assuntos
Proteoma , Espermatozoides , Animais , Masculino , Camundongos , Axonema/química , Microscopia Crioeletrônica/métodos , Flagelos/metabolismo , Microtúbulos/metabolismo , Sêmen , Espermatozoides/química , Proteoma/análiseRESUMO
Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. VIDEO ABSTRACT.
Assuntos
Doença de Alzheimer , Proteoma , Camundongos , Humanos , Animais , Proteoma/análise , Proteômica/métodos , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides , Espectrometria de Massas , Placa AmiloideRESUMO
Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete description of specific immune biomarkers. We present here a comprehensive multi-omic blood atlas for patients with varying COVID-19 severity in an integrated comparison with influenza and sepsis patients versus healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity involved cells, their inflammatory mediators and networks, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism, and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Systems-based integrative analyses including tensor and matrix decomposition of all modalities revealed feature groupings linked with severity and specificity compared to influenza and sepsis. Our approach and blood atlas will support future drug development, clinical trial design, and personalized medicine approaches for COVID-19.
Assuntos
Biomarcadores/sangue , COVID-19/patologia , Proteoma/análise , Adulto , Proteínas Sanguíneas/metabolismo , COVID-19/sangue , COVID-19/virologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Feminino , Humanos , Influenza Humana/sangue , Influenza Humana/patologia , Linfócitos/imunologia , Linfócitos/metabolismo , Aprendizado de Máquina , Masculino , Pessoa de Meia-Idade , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Análise de Componente Principal , SARS-CoV-2/isolamento & purificação , Sepse/sangue , Sepse/patologia , Índice de Gravidade de Doença , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.
Assuntos
Biomarcadores Tumorais/metabolismo , Vesículas Extracelulares/metabolismo , Neoplasias/diagnóstico , Animais , Biomarcadores Tumorais/sangue , Linhagem Celular , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Aprendizado de Máquina , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Neoplasias/metabolismo , Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos , Sensibilidade e Especificidade , Tetraspanina 29/metabolismo , Proteínas rap de Ligação ao GTP/metabolismoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is typically very mild and often asymptomatic in children. A complication is the rare multisystem inflammatory syndrome in children (MIS-C) associated with COVID-19, presenting 4-6 weeks after infection as high fever, organ dysfunction, and strongly elevated markers of inflammation. The pathogenesis is unclear but has overlapping features with Kawasaki disease suggestive of vasculitis and a likely autoimmune etiology. We apply systems-level analyses of blood immune cells, cytokines, and autoantibodies in healthy children, children with Kawasaki disease enrolled prior to COVID-19, children infected with SARS-CoV-2, and children presenting with MIS-C. We find that the inflammatory response in MIS-C differs from the cytokine storm of severe acute COVID-19, shares several features with Kawasaki disease, but also differs from this condition with respect to T cell subsets, interleukin (IL)-17A, and biomarkers associated with arterial damage. Finally, autoantibody profiling suggests multiple autoantibodies that could be involved in the pathogenesis of MIS-C.
Assuntos
Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Síndrome de Resposta Inflamatória Sistêmica/patologia , Autoanticorpos/sangue , Betacoronavirus/isolamento & purificação , COVID-19 , Criança , Pré-Escolar , Infecções por Coronavirus/complicações , Infecções por Coronavirus/virologia , Citocinas/metabolismo , Feminino , Humanos , Imunidade Humoral , Lactente , Masculino , Síndrome de Linfonodos Mucocutâneos/complicações , Síndrome de Linfonodos Mucocutâneos/imunologia , Síndrome de Linfonodos Mucocutâneos/patologia , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/virologia , Análise de Componente Principal , Proteoma/análise , SARS-CoV-2 , Índice de Gravidade de Doença , Síndrome de Resposta Inflamatória Sistêmica/etiologia , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Mitochondria contain about 1,000-1,500 proteins that fulfil multiple functions. Mitochondrial proteins originate from two genomes: mitochondrial and nuclear. Hence, proper mitochondrial function requires synchronization of gene expression in the nucleus and in mitochondria and necessitates efficient import of mitochondrial proteins into the organelle from the cytosol. Furthermore, the mitochondrial proteome displays high plasticity to allow the adaptation of mitochondrial function to cellular requirements. Maintenance of this complex and adaptable mitochondrial proteome is challenging, but is of crucial importance to cell function. Defects in mitochondrial proteostasis lead to proteotoxic insults and eventually cell death. Different quality control systems monitor the mitochondrial proteome. The cytosolic ubiquitin-proteasome system controls protein transport across the mitochondrial outer membrane and removes damaged or mislocalized proteins. Concomitantly, a number of mitochondrial chaperones and proteases govern protein folding and degrade damaged proteins inside mitochondria. The quality control factors also regulate processing and turnover of native proteins to control protein import, mitochondrial metabolism, signalling cascades, mitochondrial dynamics and lipid biogenesis, further ensuring proper function of mitochondria. Thus, mitochondrial protein quality control mechanisms are of pivotal importance to integrate mitochondria into the cellular environment.
Assuntos
Lipogênese , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Proteoma/metabolismo , Animais , Humanos , Transporte Proteico , Proteoma/análiseRESUMO
Quantitative mass spectrometry has established proteome-wide regulation of protein abundance and post-translational modifications in various biological processes. Here, we used quantitative mass spectrometry to systematically analyze the thermal stability and solubility of proteins on a proteome-wide scale during the eukaryotic cell cycle. We demonstrate pervasive variation of these biophysical parameters with most changes occurring in mitosis and G1. Various cellular pathways and components vary in thermal stability, such as cell-cycle factors, polymerases, and chromatin remodelers. We demonstrate that protein thermal stability serves as a proxy for enzyme activity, DNA binding, and complex formation in situ. Strikingly, a large cohort of intrinsically disordered and mitotically phosphorylated proteins is stabilized and solubilized in mitosis, suggesting a fundamental remodeling of the biophysical environment of the mitotic cell. Our data represent a rich resource for cell, structural, and systems biologists interested in proteome regulation during biological transitions.
Assuntos
Ciclo Celular , DNA/análise , Proteoma/análise , Proteômica/métodos , Montagem e Desmontagem da Cromatina , Análise por Conglomerados , Células HeLa , Temperatura Alta , Humanos , Espectrometria de Massas , Mitose , Fosforilação , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , RNA Polimerase II/metabolismo , SolubilidadeRESUMO
Protein degradation plays important roles in biological processes and is tightly regulated. Further, targeted proteolysis is an emerging research tool and therapeutic strategy. However, proteome-wide technologies to investigate the causes and consequences of protein degradation in biological systems are lacking. We developed "multiplexed proteome dynamics profiling" (mPDP), a mass-spectrometry-based approach combining dynamic-SILAC labeling with isobaric mass tagging for multiplexed analysis of protein degradation and synthesis. In three proof-of-concept studies, we uncover different responses induced by the bromodomain inhibitor JQ1 versus a JQ1 proteolysis targeting chimera; we elucidate distinct modes of action of estrogen receptor modulators; and we comprehensively classify HSP90 clients based on their requirement for HSP90 constitutively or during synthesis, demonstrating that constitutive HSP90 clients have lower thermal stability than non-clients, have higher affinity for the chaperone, vary between cell types, and change upon external stimuli. These findings highlight the potential of mPDP to identify dynamically controlled degradation mechanisms in cellular systems.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Proteoma/análise , Proteômica/métodos , Azepinas/química , Azepinas/metabolismo , Azepinas/farmacologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados , Estradiol/farmacologia , Humanos , Marcação por Isótopo , Células Jurkat , Células MCF-7 , Proteínas de Neoplasias/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Proteólise/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Espectrometria de Massas em Tandem , Triazóis/química , Triazóis/metabolismo , Triazóis/farmacologiaRESUMO
Osmotic diarrhea is a prevalent condition in humans caused by food intolerance, malabsorption, and widespread laxative use. Here, we assess the resilience of the gut ecosystem to osmotic perturbation at multiple length and timescales using mice as model hosts. Osmotic stress caused reproducible extinction of highly abundant taxa and expansion of less prevalent members in human and mouse microbiotas. Quantitative imaging revealed decimation of the mucus barrier during osmotic perturbation, followed by recovery. The immune system exhibited temporary changes in cytokine levels and a lasting IgG response against commensal bacteria. Increased osmolality prevented growth of commensal strains in vitro, revealing one mechanism contributing to extinction. Environmental availability of microbiota members mitigated extinction events, demonstrating how species reintroduction can affect community resilience. Our findings (1) demonstrate that even mild osmotic diarrhea can cause lasting changes to the microbiota and host and (2) lay the foundation for interventions that increase system-wide resilience.
Assuntos
Diarreia/patologia , Microbioma Gastrointestinal/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Animais , Bacteroidetes/efeitos dos fármacos , Bacteroidetes/genética , Bacteroidetes/isolamento & purificação , Ceco/química , Ceco/metabolismo , Ceco/microbiologia , Ceco/patologia , Colo/química , Colo/microbiologia , Colo/patologia , Citocinas/metabolismo , Diarreia/imunologia , Diarreia/microbiologia , Diarreia/veterinária , Fezes/microbiologia , Glicosídeo Hidrolases/metabolismo , Humanos , Imunidade Humoral/efeitos dos fármacos , Imunoglobulina G/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Metagenômica , Camundongos , Concentração Osmolar , Polietilenoglicóis/metabolismo , Proteoma/análise , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , Verrucomicrobia/efeitos dos fármacos , Verrucomicrobia/genética , Verrucomicrobia/isolamento & purificaçãoRESUMO
The transcription factor NRF2 is a master regulator of the cellular antioxidant response, and it is often genetically activated in non-small-cell lung cancers (NSCLCs) by, for instance, mutations in the negative regulator KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells. Principal among these is NR0B1, an atypical orphan nuclear receptor that we show engages in a multimeric protein complex to regulate the transcriptional output of KEAP1-mutant NSCLC cells. We further identify small molecules that covalently target a conserved cysteine within the NR0B1 protein interaction domain, and we demonstrate that these compounds disrupt NR0B1 complexes and impair the anchorage-independent growth of KEAP1-mutant cancer cells. Our findings designate NR0B1 as a druggable transcriptional regulator that supports NRF2-dependent lung cancers.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/química , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/química , Neoplasias Pulmonares/genética , Proteoma/análise , Transcriptoma , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Cisteína/metabolismo , Receptor Nuclear Órfão DAX-1/metabolismo , Redes Reguladoras de Genes , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Ligantes , Neoplasias Pulmonares/metabolismoRESUMO
We used clinical tissue from lethal metastatic castration-resistant prostate cancer (CRPC) patients obtained at rapid autopsy to evaluate diverse genomic, transcriptomic, and phosphoproteomic datasets for pathway analysis. Using Tied Diffusion through Interacting Events (TieDIE), we integrated differentially expressed master transcriptional regulators, functionally mutated genes, and differentially activated kinases in CRPC tissues to synthesize a robust signaling network consisting of druggable kinase pathways. Using MSigDB hallmark gene sets, six major signaling pathways with phosphorylation of several key residues were significantly enriched in CRPC tumors after incorporation of phosphoproteomic data. Individual autopsy profiles developed using these hallmarks revealed clinically relevant pathway information potentially suitable for patient stratification and targeted therapies in late stage prostate cancer. Here, we describe phosphorylation-based cancer hallmarks using integrated personalized signatures (pCHIPS) that shed light on the diversity of activated signaling pathways in metastatic CRPC while providing an integrative, pathway-based reference for drug prioritization in individual patients.
Assuntos
Fosfoproteínas/análise , Neoplasias de Próstata Resistentes à Castração/química , Proteoma/análise , Algoritmos , Humanos , Masculino , Medicina de Precisão , Neoplasias de Próstata Resistentes à Castração/metabolismo , Transdução de Sinais , TranscriptomaRESUMO
While alternative splicing is known to diversify the functional characteristics of some genes, the extent to which protein isoforms globally contribute to functional complexity on a proteomic scale remains unknown. To address this systematically, we cloned full-length open reading frames of alternatively spliced transcripts for a large number of human genes and used protein-protein interaction profiling to functionally compare hundreds of protein isoform pairs. The majority of isoform pairs share less than 50% of their interactions. In the global context of interactome network maps, alternative isoforms tend to behave like distinct proteins rather than minor variants of each other. Interaction partners specific to alternative isoforms tend to be expressed in a highly tissue-specific manner and belong to distinct functional modules. Our strategy, applicable to other functional characteristics, reveals a widespread expansion of protein interaction capabilities through alternative splicing and suggests that many alternative "isoforms" are functionally divergent (i.e., "functional alloforms").
Assuntos
Processamento Alternativo , Isoformas de Proteínas/metabolismo , Proteoma/metabolismo , Animais , Clonagem Molecular , Evolução Molecular , Humanos , Modelos Moleculares , Fases de Leitura Aberta , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Proteoma/análiseRESUMO
Oncogenic mutations regulate signaling within both tumor cells and adjacent stromal cells. Here, we show that oncogenic KRAS (KRAS(G12D)) also regulates tumor cell signaling via stromal cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRAS(G12D) signaling in pancreatic ductal adenocarcinoma (PDA) cells. Tumor cell KRAS(G12D) engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRAS(G12D). Consequently, reciprocal KRAS(G12D) produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRAS(G12D) alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer. VIDEO ABSTRACT.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Animais , Comunicação Celular , Humanos , Camundongos , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Proteoma/análise , Proteoma/metabolismo , Células Estromais/metabolismoRESUMO
Local mRNA translation mediates the adaptive responses of axons to extrinsic signals, but direct evidence that it occurs in mammalian CNS axons in vivo is scant. We developed an axon-TRAP-RiboTag approach in mouse that allows deep-sequencing analysis of ribosome-bound mRNAs in the retinal ganglion cell axons of the developing and adult retinotectal projection in vivo. The embryonic-to-postnatal axonal translatome comprises an evolving subset of enriched genes with axon-specific roles, suggesting distinct steps in axon wiring, such as elongation, pruning, and synaptogenesis. Adult axons, remarkably, have a complex translatome with strong links to axon survival, neurotransmission, and neurodegenerative disease. Translationally co-regulated mRNA subsets share common upstream regulators, and sequence elements generated by alternative splicing promote axonal mRNA translation. Our results indicate that intricate regulation of compartment-specific mRNA translation in mammalian CNS axons supports the formation and maintenance of neural circuits in vivo.
Assuntos
Axônios/metabolismo , Biossíntese de Proteínas , Proteoma/metabolismo , Células Ganglionares da Retina/metabolismo , Processamento Alternativo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Proteoma/análise , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Colículos Superiores/metabolismo , Transmissão SinápticaRESUMO
Stress granules are mRNA-protein granules that form when translation initiation is limited, and they are related to pathological granules in various neurodegenerative diseases. Super-resolution microscopy reveals stable substructures, referred to as cores, within stress granules that can be purified. Proteomic analysis of stress granule cores reveals a dense network of protein-protein interactions and links between stress granules and human diseases and identifies ATP-dependent helicases and protein remodelers as conserved stress granule components. ATP is required for stress granule assembly and dynamics. Moreover, multiple ATP-driven machines affect stress granules differently, with the CCT complex inhibiting stress granule assembly, while the MCM and RVB complexes promote stress granule persistence. Our observations suggest that stress granules contain a stable core structure surrounded by a dynamic shell with assembly, disassembly, and transitions between the core and shell modulated by numerous protein and RNA remodeling complexes.
Assuntos
Adenosina Trifosfatases/metabolismo , Grânulos Citoplasmáticos/química , Proteoma/análise , Ribonucleoproteínas/metabolismo , Animais , Proteínas Reguladoras de Apoptose/análise , Linhagem Celular Tumoral , Grânulos Citoplasmáticos/metabolismo , RNA Helicases DEAD-box/análise , Humanos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Proteoma/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas Repressoras/análise , Proteínas de Saccharomyces cerevisiae/análise , Azida Sódica/farmacologia , Leveduras/citologiaRESUMO
Proteomics has proved invaluable in generating large-scale quantitative data; however, the development of systems approaches for examining the proteome in vivo has lagged behind. To evaluate protein abundance and localization on a proteome scale, we exploited the yeast GFP-fusion collection in a pipeline combining automated genetics, high-throughput microscopy, and computational feature analysis. We developed an ensemble of binary classifiers to generate localization data from single-cell measurements and constructed maps of â¼3,000 proteins connected to 16 localization classes. To survey proteome dynamics in response to different chemical and genetic stimuli, we measure proteome-wide abundance and localization and identified changes over time. We analyzed >20 million cells to identify dynamic proteins that redistribute among multiple localizations in hydroxyurea, rapamycin, and in an rpd3Δ background. Because our localization and abundance data are quantitative, they provide the opportunity for many types of comparative studies, single cell analyses, modeling, and prediction. VIDEO ABSTRACT.
Assuntos
Proteoma/análise , Proteínas de Saccharomyces cerevisiae/análise , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Máquina de Vetores de Suporte , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Célula ÚnicaRESUMO
Lipids play central roles in physiology and disease, where their structural, metabolic, and signaling functions often arise from interactions with proteins. Here, we describe a set of lipid-based chemical proteomic probes and their global interaction map in mammalian cells. These interactions involve hundreds of proteins from diverse functional classes and frequently occur at sites of drug action. We determine the target profiles for several drugs across the lipid-interaction proteome, revealing that its ligandable content extends far beyond traditionally defined categories of druggable proteins. In further support of this finding, we describe a selective ligand for the lipid-binding protein nucleobindin-1 (NUCB1) and show that this compound perturbs the hydrolytic and oxidative metabolism of endocannabinoids in cells. The described chemical proteomic platform thus provides an integrated path to both discover and pharmacologically characterize a wide range of proteins that participate in lipid pathways in cells.
Assuntos
Metabolismo dos Lipídeos , Proteínas/análise , Proteínas/metabolismo , Animais , Proteínas de Ligação ao Cálcio/análise , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/análise , Avaliação Pré-Clínica de Medicamentos , Eicosanoides/metabolismo , Endocanabinoides/metabolismo , Células HEK293 , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Proteínas do Tecido Nervoso/análise , Nucleobindinas , Proteoma/análise , Proteoma/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
The availability of diverse genomes makes it possible to predict gene function based on shared evolutionary history. This approach can be challenging, however, for pathways whose components do not exhibit a shared history but rather consist of distinct "evolutionary modules." We introduce a computational algorithm, clustering by inferred models of evolution (CLIME), which inputs a eukaryotic species tree, homology matrix, and pathway (gene set) of interest. CLIME partitions the gene set into disjoint evolutionary modules, simultaneously learning the number of modules and a tree-based evolutionary history that defines each module. CLIME then expands each module by scanning the genome for new components that likely arose under the inferred evolutionary model. Application of CLIME to â¼1,000 annotated human pathways and to the proteomes of yeast, red algae, and malaria reveals unanticipated evolutionary modularity and coevolving components. CLIME is freely available and should become increasingly powerful with the growing wealth of eukaryotic genomes.
Assuntos
Algoritmos , Análise por Conglomerados , Filogenia , Humanos , Mitocôndrias/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteoma/análise , Rodófitas/genética , Rodófitas/metabolismo , Transdução de Sinais , Leveduras/genética , Leveduras/metabolismoRESUMO
The manner by which genotype and environment affect complex phenotypes is one of the fundamental questions in biology. In this study, we quantified the transcriptome--a subset of the metabolome--and, using targeted proteomics, quantified a subset of the liver proteome from 40 strains of the BXD mouse genetic reference population on two diverse diets. We discovered dozens of transcript, protein, and metabolite QTLs, several of which linked to metabolic phenotypes. Most prominently, Dhtkd1 was identified as a primary regulator of 2-aminoadipate, explaining variance in fasted glucose and diabetes status in both mice and humans. These integrated molecular profiles also allowed further characterization of complex pathways, particularly the mitochondrial unfolded protein response (UPR(mt)). UPR(mt) shows strikingly variant responses at the transcript and protein level that are remarkably conserved among C. elegans, mice, and humans. Overall, these examples demonstrate the value of an integrated multilayered omics approach to characterize complex metabolic phenotypes.
Assuntos
Perfilação da Expressão Gênica , Fígado/química , Camundongos/metabolismo , Mitocôndrias/química , Proteoma/análise , Soro/química , Animais , Glucose/metabolismo , Humanos , Cetona Oxirredutases/metabolismo , Fígado/citologia , Fígado/metabolismo , Camundongos/classificação , Camundongos/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Mitocôndrias/metabolismo , Locos de Características Quantitativas , Soro/metabolismo , Resposta a Proteínas não DobradasRESUMO
When exposed to proteotoxic environmental conditions, mammalian cells activate the cytosolic stress response in order to restore protein homeostasis. A key feature of this response is the heat shock transcription factor 1 (HSF1)-dependent expression of molecular chaperones. Here, we describe the results of an RNA interference screen in HeLa cells to identify modulators of stress response induction and attenuation. The modulator proteins are localized in multiple cellular compartments, with chromatin modifiers and nuclear protein quality control playing a central regulatory role. We find that the acetyltransferase, EP300, controls the cellular level of activatable HSF1. This involves acetylation of HSF1 at multiple lysines not required for function and results in stabilization of HSF1 against proteasomal turnover. Acetylation of functionally critical lysines during stress serves to fine-tune HSF1 activation. Finally, the nuclear proteasome system functions in attenuating the stress response by degrading activated HSF1 in a manner linked with the clearance of misfolded proteins.