RESUMEN
We describe a next-generation Drosophila protein interaction map-"DPIM2"-established from affinity purification-mass spectrometry of 5,805 baits, covering the largest fraction of the Drosophila proteome. The network contains 32,668 interactions among 3,644 proteins, organized into 632 clusters representing putative functional modules. Our analysis expands the pool of known protein interactions in Drosophila, provides annotation for poorly studied genes, and postulates previously undescribed protein interaction relationships. The predictive power and functional relevance of this network are probed through the lens of the Notch signaling pathway, and we find that newly identified members of complexes that include known Notch modifiers can also modulate Notch signaling. DPIM2 allows direct comparisons with a recently published human protein interaction network, defining the existence of functional interactions conserved across species. Thus, DPIM2 defines a valuable resource for predicting protein co-complex memberships and functional associations as well as generates functional hypotheses regarding specific protein interactions.
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Neurofilament light (NfL) concentration in cerebrospinal fluid (CSF) and blood serves as an important biomarker in neurology drug development. Changes in NfL are generally assumed to reflect changes in neuronal damage, while little is known about the clearance of NfL from biofluids. We observed an NfL increase of 3.5-fold in plasma and 5.7-fold in CSF in an asymptomatic individual at risk for genetic prion disease following 6 weeks' treatment with oral minocycline for a dermatologic indication. Other biomarkers remained normal, and proteomic analysis of CSF revealed that the spike was exquisitely specific to neurofilaments. NfL dropped nearly to normal levels 5 weeks after minocycline cessation, and the individual remained free of disease 2 years later. Plasma NfL in dermatology patients was not elevated above normal controls. Dramatically high plasma NfL (>500 pg/mL) was variably observed in some hospitalized individuals receiving minocycline. In mice, treatment with minocycline resulted in variable increases of 1.3- to 4.0-fold in plasma NfL, with complete washout 2 weeks after cessation. In neuron-microglia co-cultures, minocycline increased NfL concentration in conditioned media by 3.0-fold without any visually obvious impact on neuronal health. We hypothesize that minocycline does not cause or exacerbate neuronal damage, but instead impacts the clearance of NfL from biofluids, a potential confounder for interpretation of this biomarker.
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Scaffold proteins modulate signalling pathway activity spatially and temporally. In budding yeast, the scaffold Bem1 contributes to polarity axis establishment by regulating the GTPase Cdc42. Although different models have been proposed for Bem1 function, there is little direct evidence for an underlying mechanism. Here, we find that Bem1 directly augments the guanine exchange factor (GEF) activity of Cdc24. Bem1 also increases GEF phosphorylation by the p21-activated kinase (PAK), Cla4. Phosphorylation abrogates the scaffold-dependent stimulation of GEF activity, rendering Cdc24 insensitive to additional Bem1. Thus, Bem1 stimulates GEF activity in a reversible fashion, contributing to signalling flux through Cdc42. The contribution of Bem1 to GTPase dynamics was borne-out by in vivo imaging: active Cdc42 was enriched at the cell pole in hypophosphorylated cdc24 mutants, while hyperphosphorylated cdc24 mutants that were resistant to scaffold stimulation displayed a deficit in active Cdc42 at the pole. These findings illustrate the self-regulatory properties that scaffold proteins confer on signalling pathways.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Canales de Cloruro/metabolismo , Microscopía Intravital , Microscopía , Saccharomyces cerevisiae/fisiología , Transducción de SeñalRESUMEN
Targeted mass spectrometry assays for protein quantitation monitor peptide surrogates, which are easily multiplexed to target many peptides in a single assay. However, these assays have generally not taken advantage of sample multiplexing, which allows up to ten analyses to occur in parallel. We present a two-dimensional multiplexing workflow that utilizes synthetic peptides for each protein to prompt the simultaneous quantification of >100 peptides from up to ten mixed sample conditions. We demonstrate that targeted analysis of unfractionated lysates (2 hr) accurately reproduces the quantification of fractionated lysates (72 hr analysis) while obviating the need for peptide detection prior to quantification. We targeted 131 peptides corresponding to 69 proteins across all 60 National Cancer Institute cell lines in biological triplicate, analyzing 180 samples in only 48 hr (the equivalent of 16 min/sample). These data further elucidated a correlation between the expression of key proteins and their cellular response to drug treatment.
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Ensayos Analíticos de Alto Rendimiento , Espectrometría de Masas , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteoma , Proteómica/métodos , Antibióticos Antineoplásicos/farmacología , Biomarcadores/metabolismo , Línea Celular Tumoral , Doxorrubicina/farmacología , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Factores de Tiempo , Factores de Transcripción/metabolismo , Flujo de TrabajoRESUMEN
Mammary epithelial cells transition between periods of proliferation and quiescence during development, menstrual cycles, and pregnancy, and as a result of oncogenic transformation. Utilizing an organotypic 3D tissue culture model coupled with quantitative metabolomics and proteomics, we identified significant differences in glutamate utilization between proliferating and quiescent cells. Relative to quiescent cells, proliferating cells catabolized more glutamate via transaminases to couple non-essential amino acid (NEAA) synthesis to α-ketoglutarate generation and tricarboxylic acid (TCA) cycle anaplerosis. As cells transitioned to quiescence, glutamine consumption and transaminase expression were reduced, while glutamate dehydrogenase (GLUD) was induced, leading to decreased NEAA synthesis. Highly proliferative human tumors display high transaminase and low GLUD expression, suggesting that proliferating cancer cells couple glutamine consumption to NEAA synthesis to promote biosynthesis. These findings describe a competitive and partially redundant relationship between transaminases and GLUD, and they reveal how coupling of glutamate-derived carbon and nitrogen metabolism can be regulated to support cell proliferation.
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Células Epiteliales/citología , Células Epiteliales/metabolismo , Ácido Glutámico/metabolismo , Glándulas Mamarias Humanas/citología , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Proliferación Celular , Células Cultivadas , Femenino , Glutamato Deshidrogenasa/metabolismo , Humanos , Metabolómica , Modelos Biológicos , Isótopos de Nitrógeno , Fosfatidilinositol 3-Quinasas/metabolismo , Proteómica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Transaminasas/metabolismoRESUMEN
Isobaric labeling strategies for mass spectrometry-based proteomics enable multiplexed simultaneous quantification of samples and therefore substantially increase the sample throughput in proteomics. However, despite these benefits, current limits to multiplexing capacity are prohibitive for large sample sizes and impose limitations on experimental design. Here, we introduce a novel mechanism for increasing the multiplexing density of isobaric reagents. We present Combinatorial Isobaric Mass Tags (CMTs), an isobaric labeling architecture with the unique ability to generate multiple series of reporter ions simultaneously. We demonstrate that utilization of multiple reporter ion series improves multiplexing capacity of CMT with respect to a commercially available isobaric labeling reagent with preserved quantitative accuracy and depth of coverage in complex mixtures. We provide a blueprint for the realization of 16-plex reagents with 1 Da spacing between reporter ions and up to 28-plex at 6 mDa spacing using only 5 heavy isotopes per reagent. We anticipate that this improvement in multiplexing capacity will further advance the application of quantitative proteomics, particularly in high-throughput screening assays.
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Espectrometría de Masas/métodos , Péptidos/análisis , Proteómica/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Indicadores y Reactivos/química , Iones/químicaRESUMEN
BCL-2 is a negative regulator of apoptosis implicated in homeostatic and pathologic cell survival. The canonical anti-apoptotic mechanism involves entrapment of activated BAX by a groove on BCL-2, preventing BAX homo-oligomerization and mitochondrial membrane poration. The BCL-2 BH4 domain also confers anti-apoptotic functionality, but the mechanism is unknown. We find that a synthetic α-helical BH4 domain binds to BAX with nanomolar affinity and independently inhibits the conformational activation of BAX. Hydrogen-deuterium exchange mass spectrometry demonstrated that the N-terminal conformational changes in BAX induced by a triggering BIM BH3 helix were suppressed by the BCL-2 BH4 helix. Structural analyses localized the BH4 interaction site to a groove formed by residues of α1, α1-α2 loop, and α2-α3 and α5-α6 hairpins on the BAX surface. These data reveal a previously unappreciated binding site for targeted inhibition of BAX and suggest that the BCL-2 BH4 domain may participate in apoptosis blockade by a noncanonical interaction mechanism.
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Apoptosis , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteína X Asociada a bcl-2/química , Secuencia de Aminoácidos , Sitios de Unión/genética , Medición de Intercambio de Deuterio/métodos , Células HeLa , Humanos , Espectrometría de Masas/métodos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Conformación Proteica , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Protein interactions dictate a myriad of cellular activities that maintain health or cause disease. Dissecting these binding partnerships, and especially their sites of interaction, fuels the discovery of signaling pathways, disease mechanisms, and next-generation therapeutics. We previously applied all-hydrocarbon peptide stapling to chemically restore α-helical shape to bioactive motifs that become unfolded when taken out of context from native signaling proteins. For example, we developed stabilized alpha-helices of BCL-2 domains (SAHBs) to dissect and target protein interactions of the BCL-2 family, a critical network that regulates the apoptotic pathway. SAHBs are α-helical surrogates that bind both stable and transient physiologic interactors and have effectively uncovered novel sites of BCL-2 family protein interaction. To leverage stapled peptides for proteomic discovery, we describe our conversion of SAHBs into photoreactive agents that irreversibly capture their protein targets and facilitate rapid identification of the peptide helix binding sites. We envision that the development of photoreactive stapled peptides will accelerate the discovery of novel and unanticipated protein interactions and how they impact health and disease.
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Espectrometría de Masas/métodos , Péptidos/metabolismo , Fármacos Fotosensibilizantes/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis , Sitios de Unión , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/análisis , Fármacos Fotosensibilizantes/análisis , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteómica , Proteínas Proto-Oncogénicas c-bcl-2/análisisRESUMEN
RNA is a central component of gene-silencing pathways that regulate diverse cellular processes. In the fission yeast Schizosaccharomyces pombe, an RNA-based mechanism represses meiotic gene expression during vegetative growth. This pathway depends on the zinc finger protein Red1, which is required to degrade meiotic mRNAs as well as to target histone H3 lysine 9 (H3K9) methylation, a repressive chromatin mark, to a subset of meiotic genes. However, the mechanism of Red1 function is unknown. Here we use affinity purification and mass spectrometry to identify a Red1-containing nuclear RNA silencing (NURS) complex. In addition to Red1, this complex includes the Mtl1, Red5, Ars2, Rmn1, and Iss10 proteins and associates with several other complexes that are involved in either signaling or mediating RNA silencing. By analyzing the effects of gene knockouts and inducible knockdown alleles, we show that NURS subunits regulate RNA degradation and H3K9 methylation at meiotic genes. We also identify roles for individual NURS subunits in interactions with Mmi1, an RNA-binding protein that marks meiotic RNAs for destruction, and the nuclear exosome RNA degradation complex. Finally, we show that the levels of H3K9 methylation at meiotic genes are not sufficient to restrict RNA polymerase II access or repress gene expression during vegetative growth. Our results demonstrate that Red1 partners with other proteins to silence meiotic gene expression at the post-transcriptional level. Conservation of a NURS-like complex in human cells suggests that this pathway plays an ancient and fundamental role in RNA silencing.
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Meiosis/genética , Interferencia de ARN/fisiología , ARN Nuclear/genética , Proteínas Portadoras/genética , Cromatina/genética , Exosomas/genética , Regulación Fúngica de la Expresión Génica/genética , Histonas/genética , Metilación , ARN Polimerasa II/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , Proteínas de Schizosaccharomyces pombe/genética , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
Glucokinase (GK) is a glucose-phosphorylating enzyme that regulates insulin release and hepatic metabolism, and its loss of function is implicated in diabetes pathogenesis. GK activators (GKAs) are attractive therapeutics in diabetes; however, clinical data indicate that their benefits can be offset by hypoglycemia, owing to marked allosteric enhancement of the enzyme's glucose affinity. We show that a phosphomimetic of the BCL-2 homology 3 (BH3) α-helix derived from human BAD, a GK-binding partner, increases the enzyme catalytic rate without dramatically changing glucose affinity, thus providing a new mechanism for pharmacologic activation of GK. Remarkably, BAD BH3 phosphomimetic mediates these effects by engaging a new region near the enzyme's active site. This interaction increases insulin secretion in human islets and restores the function of naturally occurring human GK mutants at the active site. Thus, BAD phosphomimetics may serve as a new class of GKAs.
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Glucoquinasa/metabolismo , Fosfoproteínas/fisiología , Regulación Alostérica , Animales , Activación Enzimática , HumanosRESUMEN
PUMA is a proapoptotic BCL-2 family member that drives the apoptotic response to a diversity of cellular insults. Deciphering the spectrum of PUMA interactions that confer its context-dependent proapoptotic properties remains a high priority goal. Here, we report the synthesis of PUMA SAHBs, structurally stabilized PUMA BH3 helices that, in addition to broadly targeting antiapoptotic proteins, directly bind to proapoptotic BAX. NMR, photocrosslinking, and biochemical analyses revealed that PUMA SAHBs engage an α1/α6 trigger site on BAX to initiate its functional activation. We further demonstrated that a cell-permeable PUMA SAHB analog induces apoptosis in neuroblastoma cells and, like expressed PUMA protein, engages BCL-2, MCL-1, and BAX. Thus, we find that PUMA BH3 is a dual antiapoptotic inhibitor and proapoptotic direct activator, and its mimetics may serve as effective pharmacologic triggers of apoptosis in resistant human cancers.
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Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Secuencia de Aminoácidos , Animales , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Activación Enzimática , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Porosidad , Estructura Terciaria de Proteína , Proteómica , Especificidad por Sustrato , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Proapoptotic B-cell lymphoma 2 (BCL-2) antagonist/killer (BAK) and BCL-2-associated X (BAX) form toxic mitochondrial pores in response to cellular stress. Whereas BAX resides predominantly in the cytosol, BAK is constitutively localized to the outer mitochondrial membrane. Select BCL-2 homology domain 3 (BH3) helices activate BAX directly by engaging an α1/α6 trigger site. The inability to express full-length BAK has hampered full dissection of its activation mechanism. Here, we report the production of full-length, monomeric BAK by mutagenesis-based solubilization of its C-terminal α-helical surface. Recombinant BAK autotranslocates to mitochondria but only releases cytochrome c upon BH3 triggering. A direct activation mechanism was explicitly demonstrated using a liposomal system that recapitulates BAK-mediated release upon addition of BH3 ligands. Photoreactive BH3 helices mapped both triggering and autointeractions to the canonical BH3-binding pocket of BAK, whereas the same ligands crosslinked to the α1/α6 site of BAX. Thus, activation of both BAK and BAX is initiated by direct BH3-interaction but at distinct trigger sites. These structural and biochemical insights provide opportunities for developing proapoptotic agents that activate the death pathway through direct but differential engagement of BAK and BAX.
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Mitocondrias Hepáticas/química , Mitocondrias Hepáticas/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/química , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Animales , Apoptosis/fisiología , Humanos , Ratones , Ratones Noqueados , Mitocondrias Hepáticas/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/química , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Although many functions and targets have been attributed to the histone and protein deacetylase SIRT1, a comprehensive analysis of SIRT1 binding proteins yielding a high-confidence interaction map has not been established. Using a comparative statistical analysis of binding partners, we have assembled a high-confidence SIRT1 interactome. Employing this method, we identified the deubiquitinating enzyme ubiquitin-specific protease 22 (USP22), a component of the deubiquitinating module (DUBm) of the SAGA transcriptional coactivating complex, as a SIRT1-interacting partner. We found that this interaction is highly specific, requires the ZnF-UBP domain of USP22, and is disrupted by the inactivating H363Y mutation within SIRT1. Moreover, we show that USP22 is acetylated on multiple lysine residues and that alteration of a single lysine (K129) within the ZnF-UBP domain is sufficient to alter interaction of the DUBm with the core SAGA complex. Furthermore, USP22-mediated recruitment of SIRT1 activity promotes the deacetylation of individual SAGA complex components. Our results indicate an important role of SIRT1-mediated deacetylation in regulating the formation of DUBm subcomplexes within the larger SAGA complex.
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Mapas de Interacción de Proteínas , Sirtuina 1/metabolismo , Tioléster Hidrolasas/metabolismo , Acetilación , Línea Celular , Células HEK293 , Humanos , Mutación , Unión Proteica , Procesamiento Proteico-Postraduccional , Interferencia de ARN , ARN Interferente Pequeño , Sirtuina 1/genética , Tioléster Hidrolasas/genética , Transactivadores/genética , Transactivadores/metabolismo , Ubiquitina Tiolesterasa , UbiquitinaciónRESUMEN
Defining protein interactions forms the basis for discovery of biological pathways, disease mechanisms, and opportunities for therapeutic intervention. To harness the robust binding affinity and selectivity of structured peptides for interactome discovery, we engineered photoreactive stapled BH3 peptide helices that covalently capture their physiologic BCL-2 family targets. The crosslinking α helices covalently trap both static and dynamic protein interactors, and enable rapid identification of interaction sites, providing a critical link between interactome discovery and targeted drug design.
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Péptidos/química , Proteínas Proto-Oncogénicas c-bcl-2/química , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , Humanos , Datos de Secuencia Molecular , Péptidos/síntesis química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Rayos UltravioletaRESUMEN
We describe the synthesis of a fused bicyclic C-glucosylproline hybrid (GlcProH) from commercially available 2,3,4,6-tetra-O-benzyl-d-glucopyranose. The GlcProH was incorporated into the model peptides Ac-GlcProH-NHMe and Ac-Gly-GlcProH-NHMe. Postsynthetic modifications can be introduced via derivatization of the carbohydrate scaffold. Conformational analysis of the GlcProH-modified model peptides shows that while the conformation of GlcProH remains fixed, the prolyl N-terminal amide equilibrium (Kt/c) can be varied with different modifications of the carbohydrate scaffold. Simple N-acyl derivatives studied by NMR spectroscopy showed that in CD3OD there was an increase in the cis-amide content as the sugar substituents changed from benzyl (10%) to hydroxyl (22%) to acetate (36%). Similar effects were observed in DMSO-d6. The exact nature of the influence is unclear, but it most likely arises through intramolecular interactions between sugar groups and the peptidic amide backbone. Overall, our GlcProH demonstrates variation in Kt/c through tuning of the carbohydrate scaffold: a new concept in proline peptidomimetics.