RESUMO
CDK2 is a core cell-cycle kinase that phosphorylates many substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, we use several CDK2 inhibitors in clinical development to interrogate CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. Whereas CDK1 is known to compensate for loss of CDK2 in Cdk2-/- mice, this is not true of acute inhibition of CDK2. Upon CDK2 inhibition, cells exhibit a rapid loss of substrate phosphorylation that rebounds within several hours. CDK4/6 activity backstops inhibition of CDK2 and sustains the proliferative program by maintaining Rb1 hyperphosphorylation, active E2F transcription, and cyclin A2 expression, enabling re-activation of CDK2 in the presence of drug. Our results augment our understanding of CDK plasticity and indicate that co-inhibition of CDK2 and CDK4/6 may be required to suppress adaptation to CDK2 inhibitors currently under clinical assessment.
Assuntos
Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes , Animais , Camundongos , Quinases Ciclina-Dependentes/metabolismo , Ciclo Celular/fisiologia , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fosforilação , Divisão CelularRESUMO
Under fasting conditions, metazoans maintain energy balance by shifting from glucose to fat burning. In the fasted state, SIRT1 promotes catabolic gene expression by deacetylating the forkhead factor FOXO in response to stress and nutrient deprivation. The mechanisms by which hormonal signals regulate FOXO deacetylation remain unclear, however. We identified a hormone-dependent module, consisting of the Ser/Thr kinase SIK3 and the class IIa deacetylase HDAC4, which regulates FOXO activity in Drosophila. During feeding, HDAC4 is phosphorylated and sequestered in the cytoplasm by SIK3, whose activity is upregulated in response to insulin. SIK3 is inactivated during fasting, leading to the dephosphorylation and nuclear translocation of HDAC4 and to FOXO deacetylation. SIK3 mutant flies are starvation sensitive, reflecting FOXO-dependent increases in lipolysis that deplete triglyceride stores; reducing HDAC4 expression restored lipid accumulation. Our results reveal a hormone-regulated pathway that functions in parallel with the nutrient-sensing SIRT1 pathway to maintain energy balance.
Assuntos
Drosophila melanogaster/metabolismo , Metabolismo Energético , Insulina/metabolismo , Transdução de Sinais , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ingestão de Alimentos , Fatores de Transcrição Forkhead/metabolismo , Histona Desacetilases/metabolismo , Lipase/metabolismo , Metabolismo dos Lipídeos , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Triglicerídeos/metabolismoRESUMO
High-content screening for gene profiling has generally been limited to single cells. Here, we explore an alternative approach-profiling gene function by analyzing effects of gene knockdowns on the architecture of a complex tissue in a multicellular organism. We profile 554 essential C. elegans genes by imaging gonad architecture and scoring 94 phenotypic features. To generate a reference for evaluating methods for network construction, genes were manually partitioned into 102 phenotypic classes, predicting functions for uncharacterized genes across diverse cellular processes. Using this classification as a benchmark, we developed a robust computational method for constructing gene networks from high-content profiles based on a network context-dependent measure that ranks the significance of links between genes. Our analysis reveals that multi-parametric profiling in a complex tissue yields functional maps with a resolution similar to genetic interaction-based profiling in unicellular eukaryotes-pinpointing subunits of macromolecular complexes and components functioning in common cellular processes.
Assuntos
Caenorhabditis elegans/genética , Biologia Computacional/métodos , Redes Reguladoras de Genes , Técnicas Genéticas , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Embrião não Mamífero/metabolismo , Técnicas de Silenciamento de Genes , Gônadas/embriologia , FenótipoRESUMO
Tumor cells display progressive changes in metabolism that correlate with malignancy, including development of a lipogenic phenotype. How stored fats are liberated and remodeled to support cancer pathogenesis, however, remains unknown. Here, we show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in nonaggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity-phenotypes that are reversed by an MAGL inhibitor. Impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of protumorigenic signals. PAPERFLICK:
Assuntos
Ácidos Graxos/metabolismo , Monoacilglicerol Lipases/metabolismo , Neoplasias Ovarianas/metabolismo , Animais , Linhagem Celular , Feminino , Humanos , Camundongos , Monoacilglicerol Lipases/genética , Monoglicerídeos/metabolismo , Transplante de Neoplasias , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
The expansion of the target landscape of covalent inhibitors requires the engagement of nucleophiles beyond cysteine. Although the conserved catalytic lysine in protein kinases is an attractive candidate for a covalent approach, selectivity remains an obvious challenge. Moreover, few covalent inhibitors have been shown to engage the kinase catalytic lysine in animals. We hypothesized that reversible, lysine-targeted inhibitors could provide sustained kinase engagement in vivo, with selectivity driven in part by differences in residence time. By strategically linking benzaldehydes to a promiscuous kinase binding scaffold, we developed chemoproteomic probes that reversibly and covalently engage >200 protein kinases in cells and mice. Probe-kinase residence time was dramatically enhanced by a hydroxyl group ortho to the aldehyde. Remarkably, only a few kinases, including Aurora A, showed sustained, quasi-irreversible occupancy in vivo, the structural basis for which was revealed by X-ray crystallography. We anticipate broad application of salicylaldehyde-based probes to proteins that lack a druggable cysteine.
Assuntos
Lisina , Inibidores de Proteínas Quinases , Animais , Cisteína/metabolismo , Lisina/metabolismo , Camundongos , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismoRESUMO
Despite their diminutive size, islets of Langerhans play a large role in maintaining systemic energy balance in the body. New technologies have enabled us to go from studying the whole pancreas to isolated whole islets, to partial islet sections, and now to islet substructures isolated from within the islet. Using a microfluidic nanodroplet-based proteomics platform coupled with laser capture microdissection and field asymmetric waveform ion mobility spectrometry, we present an in-depth investigation of protein profiles specific to features within the islet. These features include the islet-acinar interface vascular tissue, inner islet vasculature, isolated endocrine cells, whole islet with vasculature, and acinar tissue from around the islet. Compared to interface vasculature, unique protein signatures observed in the inner vasculature indicate increased innervation and intra-islet neuron-like crosstalk. We also demonstrate the utility of these data for identifying localized structure-specific drug-target interactions using existing protein/drug binding databases.
Assuntos
Ilhotas Pancreáticas , Ilhotas Pancreáticas/metabolismo , Proteômica/métodos , Proteínas/metabolismo , Microdissecção e Captura a LaserRESUMO
Poly-ADP-ribosyltransferases play a critical role in DNA repair and cell death, and poly(ADP-ribosyl) polymerase 1 (PARP1) is a particularly important therapeutic target for the treatment of breast cancer because of its synthetic lethal relationship with breast cancer susceptibility proteins 1 and 2. Numerous PARP1 inhibitors have been developed, and their efficacy in cancer treatment is attributed to both the inhibition of enzymatic activity and their ability to trap PARP1 on to the damaged DNA, which is cytotoxic. Of the clinical PARP inhibitors, talazoparib is the most effective at trapping PARP1 on damaged DNA. Biochemically, talazoparib is also suspected to be a potent inhibitor of PARP5a/b (tankyrase1/2 [TNKS1/2]), which is an important regulator of Wnt/ß-catenin pathway. Here we show using competition experiments in cell lysate that, at a clinically relevant concentration, talazoparib can potentially bind and engage TNKS1. Using surface plasmon resonance, we measured the dissociation constants of talazoparib, olaparib, niraparib, and veliparib for their interaction with PARP1 and TNKS1. The results show that talazoparib has strong affinity for PARP1 as well as uniquely strong affinity for TNKS1. Finally, we used crystallography and hydrogen deuterium exchange mass spectroscopy to dissect the molecular mechanism of differential selectivity of these PARP1 inhibitors. From these data, we conclude that subtle differences between the ligand-binding sites of PARP1 and TNKS1, differences in the electrostatic nature of the ligands, protein dynamics, and ligand conformational energetics contribute to the different pharmacology of these PARP1 inhibitors. These results will help in the design of drugs to treat Wnt/ß-catenin pathway-related cancers, such as colorectal cancers.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Poli(ADP-Ribose) Polimerase-1/genética , Tanquirases/genética , Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Sítios de Ligação/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/genética , Feminino , Humanos , Indazóis/farmacologia , Ligantes , Ftalazinas/farmacologia , Piperidinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
BACKGROUND: Papain-like Cys Proteases (PLCPs) and Vacuolar Processing Enzymes (VPEs) are amongst the most highly expressed proteases during leaf senescence in Arabidopsis. Using activity-based protein profiling (ABPP), a method that enables detection of active enzymes within a complex sample using chemical probes, the activities of PLCPs and VPEs were investigated in individually darkened leaves of Arabidopsis, and their role in senescence was tested in null mutants. RESULTS: ABPP and mass spectrometry revealed an increased activity of several PLCPs, particularly RD21A and AALP. By contrast, despite increased VPE transcript levels, active VPE decreased in individually darkened leaves. Eight protease knock-out lines and two protease over expressing lines were subjected to senescence phenotype analysis to determine the importance of individual protease activities to senescence. Unexpectedly, despite the absence of dominating PLCP activities in these plants, the rubisco and chlorophyll decline in individually darkened leaves and the onset of whole plant senescence were unaltered. However, a significant delay in progression of whole plant senescence was observed in aalp-1 and rd21A-1/aalp-1 mutants, visible in the reduced number of senescent leaves. CONCLUSIONS: Major Cys protease activities are not essential for dark-induced and developmental senescence and only a knock out line lacking AALP shows a slight but significant delay in plant senescence.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Cisteína Proteases/metabolismo , Folhas de Planta/enzimologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Clorofila/metabolismo , Cisteína Proteases/genética , Escuridão , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/efeitos da radiação , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismoRESUMO
The network of activator protein-protein interactions (PPIs) that underpin transcription initiation is poorly defined, particularly in the cellular context. The transient nature of these contacts and the often low abundance of the participants present significant experimental hurdles. Through the coupling of in vivo covalent chemical capture and shotgun LC-MS/MS (MuDPIT) analysis, we can trap the PPIs of transcriptional activators in a cellular setting and identify the binding partners in an unbiased fashion. Using this approach, we discover that the prototypical activators Gal4 and VP16 target the Snf1 (AMPK) kinase complex via direct interactions with both the core enzymatic subunit Snf1 and the exchangeable subunit Gal83. Further, we use a tandem reversible formaldehyde and irreversible covalent chemical capture approach (TRIC) to capture the Gal4-Snf1 interaction at the Gal1 promoter in live yeast. Together, these data support a critical role for activator PPIs in both the recruitment and positioning of important enzymatic complexes at a gene promoter and represent a technical advancement in the discovery of new cellular binding targets of transcriptional activators.
Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Transativadores , Ativação TranscricionalRESUMO
Sulfonyl fluoride (SF)-based activity probes have become important tools in chemical biology. Herein, exploiting the relative chemical stability of SF to carry out a number of unprecedented SF-sparing functional group manipulations, we report the chemoselective synthesis of a toolbox of highly functionalized aryl SF monomers that we used to quickly prepare SF chemical biology probes. In addition to SF, the monomers bear an embedded click handle (a terminal alkyne that can perform copper(I)-mediated azide-alkyne cycloaddition). The monomers can be used either as fragments to prepare clickable SF analogues of drugs (biologically active compounds) bearing an aryl ring or, alternatively, attached to drugs as minimalist clickable aryl SF substituents.
Assuntos
Sondas Moleculares/síntese química , Ácidos Sulfínicos/síntese química , Química Click , Modelos Moleculares , Sondas Moleculares/química , Estrutura Molecular , Ácidos Sulfínicos/químicaRESUMO
Phenotypic screening is making a comeback in drug discovery as the maturation of chemical proteomics methods has facilitated target identification for bioactive small molecules. A limitation of these approaches is that time-consuming genetic methods or other means are often required to determine the biologically relevant target (or targets) from among multiple protein-compound interactions that are typically detected. Here, we have combined phenotypic screening of a directed small-molecule library with competitive activity-based protein profiling to map and functionally characterize the targets of screening hits. Using this approach, we identify carboxylesterase 3 (Ces3, also known as Ces1d) as a primary molecular target of bioactive compounds that promote lipid storage in adipocytes. We further show that Ces3 activity is markedly elevated during adipocyte differentiation. Treatment of two mouse models of obesity-diabetes with a Ces3 inhibitor ameliorates multiple features of metabolic syndrome, illustrating the power of the described strategy to accelerate the identification and pharmacologic validation of new therapeutic targets.
Assuntos
Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Diabetes Mellitus/genética , Obesidade/genética , Fenótipo , Bibliotecas de Moléculas Pequenas , Animais , Células Cultivadas , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Descoberta de Drogas , Camundongos , Análise Serial de Proteínas , ProteômicaRESUMO
Kinases are principal components of signal transduction pathways and the focus of intense basic and drug discovery research. Irreversible inhibitors that covalently modify non-catalytic cysteines in kinase active sites have emerged as valuable probes and approved drugs. Many protein classes, however, have functional cysteines, and therefore understanding the proteome-wide selectivity of covalent kinase inhibitors is imperative. Here, we accomplish this objective using activity-based protein profiling coupled with quantitative MS to globally map the targets, both specific and nonspecific, of covalent kinase inhibitors in human cells. Many of the specific off-targets represent nonkinase proteins that, notably, have conserved active site cysteines. We define windows of selectivity for covalent kinase inhibitors and show that, when these windows are exceeded, rampant proteome-wide reactivity and kinase target-independent cell death conjointly occur. Our findings, taken together, provide an experimental road map to illuminate opportunities and surmount challenges for the development of covalent kinase inhibitors.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteoma/genética , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisteína/química , Genes erbB-1/genética , Humanos , Cinética , Piperidinas , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
The de novo biosynthesis of purines is carried out by a highly conserved metabolic pathway that includes several validated targets for anticancer, immunosuppressant, and anti-inflammatory chemotherapeutics. The six enzymes in humans that catalyze the 10 chemical steps from phosphoribosylpyrophosphate to inosine monophosphate were recently shown to associate into a dynamic multiprotein complex called the purinosome. Here, we demonstrate that heat shock protein 90 (Hsp90), heat shock protein 70 (Hsp70), and several cochaperones functionally colocalize with this protein complex. Knockdown of expression levels of the identified cochaperones leads to disruption of purinosomes. In addition, small molecule inhibitors of Hsp90 and Hsp70 reversibly disrupt purinosomes and are shown to have a synergistic effect with methotrexate, an anticancer agent that targets purine biosynthesis. These data implicate the Hsp90/Hsp70 chaperone machinery in the assembly of the purinosome and provide a strategy for the development of improved anticancer therapies that disrupt purine biosynthesis.
Assuntos
Vias Biossintéticas/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/metabolismo , Purinas/biossíntese , Formazans , Células HeLa , Humanos , Imunoprecipitação , Luciferases , Metotrexato , Estrutura Molecular , Sais de TetrazólioRESUMO
Under fasting conditions, increases in circulating glucagon maintain glucose balance by promoting hepatic gluconeogenesis. Triggering of the cAMP pathway stimulates gluconeogenic gene expression through the PKA-mediated phosphorylation of the cAMP response element binding (CREB) protein and via the dephosphorylation of the latent cytoplasmic CREB regulated transcriptional coactivator 2 (CRTC2). CREB and CRTC2 activities are increased in insulin resistance, in which they promote hyperglycemia because of constitutive induction of the gluconeogenic program. The extent to which CREB and CRTC2 are coordinately up-regulated in response to glucagon, however, remains unclear. Here we show that, following its activation, CRTC2 enhances CREB phosphorylation through an association with the protein arginine methyltransferase 5 (PRMT5). In turn, PRMT5 was found to stimulate CREB phosphorylation via increases in histone H3 Arg2 methylation that enhanced chromatin accessibility at gluconeogenic promoters. Because depletion of PRMT5 lowers hepatic glucose production and gluconeogenic gene expression, these results demonstrate how a chromatin-modifying enzyme regulates a metabolic program through epigenetic changes that impact the phosphorylation of a transcription factor in response to hormonal stimuli.
Assuntos
Metabolismo Energético/fisiologia , Epigênese Genética/fisiologia , Jejum/fisiologia , Regulação da Expressão Gênica/fisiologia , Gluconeogênese/fisiologia , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Glicemia/metabolismo , Imunoprecipitação da Cromatina , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Eletroforese em Gel de Poliacrilamida , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Imunoprecipitação , Luciferases , Espectrometria de Massas , Metilação , Camundongos , Fosforilação , Fatores de Transcrição/metabolismoRESUMO
Infection of plants by bacterial leaf pathogens at wound sites is common in nature. Plants defend wound sites to prevent pathogen invasion, but several pathogens can overcome spatial restriction and enter leaf tissues. The molecular mechanisms used by pathogens to suppress containment at wound infection sites are poorly understood. Here, we studied Pseudomonas syringae strains causing brown spot on bean and blossom blight on pear. These strains exist as epiphytes that can cause disease upon wounding caused by hail, sand storms and frost. We demonstrate that these strains overcome spatial restriction at wound sites by producing syringolin A (SylA), a small molecule proteasome inhibitor. Consequently, SylA-producing strains are able to escape from primary infection sites and colonize adjacent tissues along the vasculature. We found that SylA diffuses from the primary infection site and suppresses acquired resistance in adjacent tissues by blocking signaling by the stress hormone salicylic acid (SA). Thus, SylA diffusion creates a zone of SA-insensitive tissue that is prepared for subsequent colonization. In addition, SylA promotes bacterial motility and suppresses immune responses at the primary infection site. These local immune responses do not affect bacterial growth and were weak compared to effector-triggered immunity. Thus, SylA facilitates colonization from wounding sites by increasing bacterial motility and suppressing SA signaling in adjacent tissues.
Assuntos
Nicotiana/microbiologia , Peptídeos Cíclicos/metabolismo , Doenças das Plantas/microbiologia , Inibidores de Proteassoma/metabolismo , Pseudomonas syringae/metabolismo , Infecção dos Ferimentos/microbiologia , Sequência de Aminoácidos , Interações Hospedeiro-Patógeno , Dados de Sequência Molecular , Proteínas de Plantas , Complexo de Endopeptidases do Proteassoma/genética , Transdução de SinaisRESUMO
The Ro 60-kDa autoantigen, a ring-shaped RNA-binding protein, traffics between the nucleus and cytoplasm in vertebrate cells. In some vertebrate nuclei, Ro binds misfolded noncoding RNAs and may function in quality control. In the cytoplasm, Ro binds noncoding RNAs called Y RNAs. Y RNA binding blocks a nuclear accumulation signal, retaining Ro in the cytoplasm. Following UV irradiation, this signal becomes accessible, allowing Ro to accumulate in nuclei. To investigate how other cellular components influence the function and subcellular location of Ro, we identified several proteins that copurify with the mouse Ro protein. Here, we report that the zipcode-binding protein ZBP1 influences the subcellular localization of both Ro and the Y3 RNA. Binding of ZBP1 to the Ro/Y3 complex increases after UV irradiation and requires the Y3 RNA. Despite the lack of an identifiable CRM1-dependent export signal, nuclear export of Ro is sensitive to the CRM1 inhibitor leptomycin B. In agreement with a previous report, we find that ZBP1 export is partly dependent on CRM1. Both Ro and Y3 RNA accumulate in nuclei when ZBP1 is depleted. Our data indicate that ZBP1 may function as an adapter to export the Ro/Y3 RNA complex from nuclei.
Assuntos
Autoantígenos/metabolismo , Núcleo Celular/metabolismo , Glicoproteínas/metabolismo , RNA Citoplasmático Pequeno/metabolismo , RNA não Traduzido/metabolismo , Ribonucleoproteínas/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Linhagem Celular , Ácidos Graxos Insaturados/farmacologia , Glicoproteínas/genética , Carioferinas/antagonistas & inibidores , Carioferinas/metabolismo , Camundongos , Proteínas de Ligação a RNA , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Raios Ultravioleta , Proteína Exportina 1RESUMO
Filamentous cyanobacteria of the genus Lyngbya are important contributors to coral reef ecosystems, occasionally forming dominant cover and impacting the health of many other co-occurring organisms. Moreover, they are extraordinarily rich sources of bioactive secondary metabolites, with 35% of all reported cyanobacterial natural products deriving from this single pantropical genus. However, the true natural product potential and life strategies of Lyngbya strains are poorly understood because of phylogenetic ambiguity, lack of genomic information, and their close associations with heterotrophic bacteria and other cyanobacteria. To gauge the natural product potential of Lyngbya and gain insights into potential microbial interactions, we sequenced the genome of Lyngbya majuscula 3L, a Caribbean strain that produces the tubulin polymerization inhibitor curacin A and the molluscicide barbamide, using a combination of Sanger and 454 sequencing approaches. Whereas â¼ 293,000 nucleotides of the draft genome are putatively dedicated to secondary metabolism, this is far too few to encode a large suite of Lyngbya metabolites, suggesting Lyngbya metabolites are strain specific and may be useful in species delineation. Our analysis revealed a complex gene regulatory network, including a large number of sigma factors and other regulatory proteins, indicating an enhanced ability for environmental adaptation or microbial associations. Although Lyngbya species are reported to fix nitrogen, nitrogenase genes were not found in the genome or by PCR of genomic DNA. Subsequent growth experiments confirmed that L. majuscula 3L is unable to fix atmospheric nitrogen. These unanticipated life history characteristics challenge current views of the genus Lyngbya.
Assuntos
Cianobactérias/genética , Cianobactérias/fisiologia , Redes Reguladoras de Genes , Genoma Bacteriano/genética , Ciclopropanos , Ecologia , Genes Bacterianos/fisiologia , Biologia Marinha , Fixação de Nitrogênio/genética , Análise de Sequência de DNA , TiazóisRESUMO
More than half of the ~20,000 protein-encoding human genes have at least one paralog. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to a subset of paralogous proteins. Here, we explore whether such covalent compound-cysteine interactions can be used to discover ligandable pockets in paralogs that lack the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we mutated the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling (ABPP) that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide-N112C-CCNE1 interaction into a NanoBRET-ABPP assay capable of identifying compounds that reversibly inhibit both N112C- and WT-CCNE1:CDK2 complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings thus provide a roadmap for leveraging electrophile-cysteine interactions to extend the ligandability of the proteome beyond covalent chemistry.
RESUMO
Papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes associated with development, immunity, and senescence. Although many properties have been described for individual proteases, the distribution of these characteristics has not been studied collectively. Here, we analyzed 723 plant PLCPs and classify them into nine subfamilies that are present throughout the plant kingdom. Analysis of these subfamilies revealed previously unreported distinct subfamily-specific functional and structural characteristics. For example, the NPIR and KDEL localization signals are distinctive for subfamilies, and the carboxyl-terminal granulin domain occurs in two PLCP subfamilies, in which some individual members probably evolved by deletion of the granulin domains. We also discovered a conserved double cysteine in the catalytic site of SAG12-like proteases and two subfamily-specific disulfides in RD19A-like proteases. Protease activity profiling of representatives of the PLCP subfamilies using novel fluorescent probes revealed striking polymorphic labeling profiles and remarkably distinct pH dependency. Competition assays with peptide-epoxide scanning libraries revealed common and unique inhibitory fingerprints. Finally, we expand the detection of PLCPs by identifying common and organ-specific protease activities and identify previously undetected proteases upon labeling with cell-penetrating probes in vivo. This study provides the plant protease research community with tools for further functional annotation of plant PLCPs.
Assuntos
Família Multigênica , Papaína/classificação , Papaína/metabolismo , Plantas/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Sequência Conservada/genética , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos/efeitos dos fármacos , Papaína/antagonistas & inibidores , Papaína/química , Mapeamento de Peptídeos , Filogenia , Progranulinas , Inibidores de Proteases/farmacologia , Coloração e RotulagemRESUMO
Serine hydrolases are a diverse enzyme class representing â¼1% of all human proteins. The biological functions of most serine hydrolases remain poorly characterized owing to a lack of selective inhibitors to probe their activity in living systems. Here we show that a substantial number of serine hydrolases can be irreversibly inactivated by 1,2,3-triazole ureas, which show negligible cross-reactivity with other protein classes. Rapid lead optimization by click chemistry-enabled synthesis and competitive activity-based profiling identified 1,2,3-triazole ureas that selectively inhibit enzymes from diverse branches of the serine hydrolase class, including peptidases (acyl-peptide hydrolase, or APEH), lipases (platelet-activating factor acetylhydrolase-2, or PAFAH2) and uncharacterized hydrolases (α,ß-hydrolase-11, or ABHD11), with exceptional potency in cells (sub-nanomolar) and mice (<1 mg kg(-1)). We show that APEH inhibition leads to accumulation of N-acetylated proteins and promotes proliferation in T cells. These data indicate 1,2,3-triazole ureas are a pharmacologically privileged chemotype for serine hydrolase inhibition, combining broad activity across the serine hydrolase class with tunable selectivity for individual enzymes.