RESUMEN
Covalent peptide binders have found applications as activity-based probes and as irreversible therapeutic inhibitors. Currently, there is no rapid, label-free, and tunable affinity selection platform to enrich covalent reactive peptide binders from synthetic libraries. We address this challenge by developing a reversibly reactive affinity selection platform termed ReAct-ASMS enabled by tandem high-resolution mass spectrometry (MS/MS) to identify covalent peptide binders to native protein targets. It uses mixed disulfide-containing peptides to build reversible peptide-protein conjugates that can enrich for covalent variants, which can be sequenced by MS/MS after reduction. Using this platform, we identified covalent peptide binders against two oncoproteins, human papillomavirus 16 early protein 6 (HPV16 E6) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 protein (Pin1). The resulting peptide binders efficiently and selectively cross-link Cys58 of E6 at 37 °C and Cys113 of Pin1 at room temperature, respectively. ReAct-ASMS enables the identification of highly selective covalent peptide binders for diverse molecular targets, introducing an applicable platform to assist preclinical therapeutic development pipelines.
Asunto(s)
Péptidos , Péptidos/química , Proteínas Oncogénicas Virales/química , Humanos , Peptidilprolil Isomerasa de Interacción con NIMA/antagonistas & inhibidores , Peptidilprolil Isomerasa de Interacción con NIMA/química , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Espectrometría de Masas en Tándem/métodos , Unión ProteicaRESUMEN
Human papillomavirus (HPV) is a significant contributor to the global cancer burden, and its carcinogenic activity is facilitated in part by the HPV early protein 6 (E6), which interacts with the E3-ligase E6AP, also known as UBE3A, to promote degradation of the tumor suppressor, p53. In this study, we present a single-particle cryoEM structure of the full-length E6AP protein in complex with HPV16 E6 (16E6) and p53, determined at a resolution of ~3.3 Å. Our structure reveals extensive protein-protein interactions between 16E6 and E6AP, explaining their picomolar binding affinity. These findings shed light on the molecular basis of the ternary complex, which has been pursued as a potential therapeutic target for HPV-driven cervical, anal, and oropharyngeal cancers over the last two decades. Understanding the structural and mechanistic underpinnings of this complex is crucial for developing effective therapies to combat HPV-induced cancers. Our findings may help to explain why previous attempts to disrupt this complex have failed to generate therapeutic modalities and suggest that current strategies should be reevaluated.
Asunto(s)
Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Humanos , Proteína p53 Supresora de Tumor/metabolismo , Papillomavirus Humano 16/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Oncogénicas Virales/genética , Genes Supresores de TumorRESUMEN
Human papillomavirus (HPV) infections account for nearly all cervical cancer cases, which is the fourth most common cancer in women worldwide. High-risk variants, including HPV16, drive tumorigenesis in part by promoting the degradation of the tumor suppressor p53. This degradation is mediated by the HPV early protein 6 (E6), which recruits the E3 ubiquitin ligase E6AP and redirects its activity towards ubiquitinating p53. Targeting the protein interaction interface between HPV E6 and E6AP is a promising modality to mitigate HPV-mediated degradation of p53. In this study, we designed a covalent peptide inhibitor, termed reactide, that mimics the E6AP LXXLL binding motif by selectively targeting cysteine 58 in HPV16 E6 with quantitative conversion. This reactide provides a starting point in the development of covalent peptidomimetic inhibitors for intervention against HPV-driven cancers.
RESUMEN
Phosphatidylinositol (PtdIns) transfer proteins (PITPs) enhance the activities of PtdIns 4-OH kinases that generate signaling pools of PtdIns-4-phosphate. In that capacity, PITPs serve as key regulators of lipid signaling in eukaryotic cells. Although the PITP phospholipid exchange cycle is the engine that stimulates PtdIns 4-OH kinase activities, the underlying mechanism is not understood. Herein, we apply an integrative structural biology approach to investigate interactions of the yeast PITP Sec14 with small-molecule inhibitors (SMIs) of its phospholipid exchange cycle. Using a combination of X-ray crystallography, solution NMR spectroscopy, and atomistic MD simulations, we dissect how SMIs compete with native Sec14 phospholipid ligands and arrest phospholipid exchange. Moreover, as Sec14 PITPs represent new targets for the development of next-generation antifungal drugs, the structures of Sec14 bound to SMIs of diverse chemotypes reported in this study will provide critical information required for future structure-based design of next-generation lead compounds directed against Sec14 PITPs of virulent fungi.
Asunto(s)
Antifúngicos , Diseño de Fármacos , Proteínas de Transferencia de Fosfolípidos , Proteínas de Saccharomyces cerevisiae , Transporte Biológico/efectos de los fármacos , Fosfatidilinositoles/metabolismo , Proteínas de Transferencia de Fosfolípidos/antagonistas & inhibidores , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Transducción de Señal , Antifúngicos/química , Antifúngicos/farmacologíaRESUMEN
Cellular senescence and the senescence-associated secretory phenotype (SASP) are implicated in aging and age-related disease, and SASP-related inflammation is thought to contribute to tissue dysfunction in aging and diseased animals. However, whether and how SASP factors influence the regenerative capacity of tissues remains unclear. Here, using intestinal organoids as a model of tissue regeneration, we show that SASP factors released by senescent fibroblasts deregulate stem cell activity and differentiation and ultimately impair crypt formation. We identify the secreted N-terminal domain of Ptk7 as a key component of the SASP that activates non-canonical Wnt / Ca2+ signaling through FZD7 in intestinal stem cells (ISCs). Changes in cytosolic [Ca2+] elicited by Ptk7 promote nuclear translocation of YAP and induce expression of YAP/TEAD target genes, impairing symmetry breaking and stem cell differentiation. Our study discovers secreted Ptk7 as a factor released by senescent cells and provides insight into the mechanism by which cellular senescence contributes to tissue dysfunction in aging and disease.
Asunto(s)
Diferenciación Celular , Proteínas Tirosina Quinasas Receptoras , Células Madre , Animales , Ratones , Envejecimiento , Diferenciación Celular/genética , Senescencia Celular/genética , Intestinos/citología , Intestinos/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Células Madre/metabolismo , Vía de Señalización Wnt , Proteínas Señalizadoras YAPRESUMEN
Developing peptide-based tools to fine-tune growth signaling pathways, in particular molecules with exquisite selectivity and high affinities, opens up opportunities for cellular reprogramming in tissue regeneration. Here, we present a library based on cystine-knot peptides (CKPs) that incorporate multiple loops for randomization and selection via directed evolution. Resulting binders could be assembled into multimeric structures to fine-tune cellular signaling. An example is presented for the Wnt pathway, which plays a key role in the homeostasis and regeneration of tissues such as lung, skin, and intestine. We discovered picomolar affinity CKP agonists of the human LPR6 receptor by exploring the limits of the topological manipulation of LRP6 dimerization. Structural analyses revealed that the agonists bind at the first ß-propeller domain of LRP6, mimicking the natural Wnt inhibitors DKK1 and SOST. However, the CKP agonists exhibit a different mode of action as they amplify the signaling of natural Wnt ligands but do not activate the pathway by themselves. In an alveolosphere organoid model, the CKP agonists induced alveolar stem cell activity. They also stimulated growth in primary human intestinal organoids. The approach described here advances the important frontier of next-generation agonist design and could be applied to other signaling pathways to discover tunable agonist ligands.
Asunto(s)
Vía de Señalización Wnt , beta Catenina , Humanos , beta Catenina/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas Wnt/metabolismo , Cistina , Ligandos , PéptidosRESUMEN
Insulin-like growth factor (IGF) signaling is highly conserved and tightly regulated by proteases including Pregnancy-Associated Plasma Protein A (PAPP-A). PAPP-A and its paralog PAPP-A2 are metalloproteases that mediate IGF bioavailability through cleavage of IGF binding proteins (IGFBPs). Here, we present single-particle cryo-EM structures of the catalytically inactive mutant PAPP-A (E483A) in complex with a peptide from its substrate IGFBP5 (PAPP-ABP5) and also in its substrate-free form, by leveraging the power of AlphaFold to generate a high quality predicted model as a starting template. We show that PAPP-A is a flexible trans-dimer that binds IGFBP5 via a 25-amino acid anchor peptide which extends into the metalloprotease active site. This unique IGFBP5 anchor peptide that mediates the specific PAPP-A-IGFBP5 interaction is not found in other PAPP-A substrates. Additionally, we illustrate the critical role of the PAPP-A central domain as it mediates both IGFBP5 recognition and trans-dimerization. We further demonstrate that PAPP-A trans-dimer formation and distal inter-domain interactions are both required for efficient proteolysis of IGFBP4, but dispensable for IGFBP5 cleavage. Together the structural and biochemical studies reveal the mechanism of PAPP-A substrate binding and selectivity.
Asunto(s)
Proteína Plasmática A Asociada al Embarazo , Somatomedinas , Aminoácidos/metabolismo , Péptidos/metabolismo , Proteína Plasmática A Asociada al Embarazo/química , Proteína Plasmática A Asociada al Embarazo/metabolismo , Unión Proteica , Somatomedinas/metabolismoRESUMEN
The emergence of fungal "superbugs" resistant to the limited cohort of anti-fungal agents available to clinicians is eroding our ability to effectively treat infections by these virulent pathogens. As the threat of fungal infection is escalating worldwide, this dwindling response capacity is fueling concerns of impending global health emergencies. These developments underscore the urgent need for new classes of anti-fungal drugs and, therefore, the identification of new targets. Phosphoinositide signaling does not immediately appear to offer attractive targets due to its evolutionary conservation across the Eukaryota. However, recent evidence argues otherwise. Herein, we discuss the evidence identifying Sec14-like phosphatidylinositol transfer proteins (PITPs) as unexplored portals through which phosphoinositide signaling in virulent fungi can be chemically disrupted with exquisite selectivity. Recent identification of lead compounds that target fungal Sec14 proteins, derived from several distinct chemical scaffolds, reveals exciting inroads into the rational design of next generation Sec14 inhibitors. Development of appropriately refined next generation Sec14-directed inhibitors promises to expand the chemical weaponry available for deployment in the shifting field of engagement between fungal pathogens and their human hosts.
Asunto(s)
Antifúngicos/farmacología , Micosis/tratamiento farmacológico , Proteínas de Transferencia de Fosfolípidos/antagonistas & inhibidores , Animales , Humanos , Micosis/metabolismoRESUMEN
Sec14, a yeast phosphatidylinositol/phosphatidylcholine transfer protein, functions at the trans-Golgi membranes. It lacks domains involved in protein-protein or protein-lipid interactions and consists solely of the Sec14 domain; hence, the mechanism underlying Sec14 function at proper sites remains unclear. In this study, we focused on the lipid packing of membranes and evaluated its association with in vitro Sec14 lipid transfer activity. Phospholipid transfer assays using pyrene-labelled phosphatidylcholine suggested that increased membrane curvature as well as the incorporation of phosphatidylethanolamine accelerated the lipid transfer. The quantity of membrane-bound Sec14 significantly increased in these membranes, indicating that "packing defects" of the membranes promote the membrane binding and phospholipid transfer of Sec14. Increased levels of phospholipid unsaturation promoted Sec14-mediated PC transfer, but had little effect on the membrane binding of the protein. Our results demonstrate the possibility that the location and function of Sec14 are regulated by the lipid packing states produced by a translocase activity at the trans-Golgi network.
Asunto(s)
Membranas Artificiales , Proteínas de Transferencia de Fosfolípidos/química , Fosfolípidos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Dominios ProteicosRESUMEN
Frizzled 7 (FZD7) receptors have been shown to play a central role in intestinal stem cell regeneration and, more recently, in Clostridium difficile pathogenesis. Yet, targeting FZD7 receptors with small ligands has not been explored as an approach to block C. difficile pathogenesis. Here, we report the discovery of high affinity peptides that selectively bind to FZD7 receptors. We describe an integrated approach for lead optimization, utilizing structure-based rational design and directed evolution, to enhance the peptide binding affinity while still maintaining FZD7 receptor selectivity. This work yielded new peptide leads with picomolar binding constants to FZD7 as measured by biophysical methods. The new peptides block the interaction between C. difficile toxin B (TcdB) and FZD receptors and perturb C. difficile pathogenesis in epithelial cells. As such, our findings provide a proof of concept that targeting FZD receptors could be a viable pharmacological approach to protect epithelial cells from TcdB pathogenicity.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/antagonistas & inhibidores , Clostridioides difficile/química , Células Epiteliales/efectos de los fármacos , Receptores Frizzled/antagonistas & inhibidores , Péptidos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Células Epiteliales/metabolismo , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , Péptidos/química , Relación Estructura-ActividadRESUMEN
Wnts are lipid-modified proteins that regulate stem cell signaling via Frizzled receptors on the cell surface. Determination of binding interactions between lipid-modified Wnt proteins and their Frizzled receptors has been challenging due to the lack of availability of facile detection methods and technical hurdles associated with generating the relevant reagents. Here we report an enzyme-linked immunosorbent assay to measure the binding of a biotinylated form of lipid-modified Wnt3a to the extracellular cysteine-rich domain of Frizzled receptor. The method described herein is robust and rapid, uses minimum volumes of reagents, and can be conducted in a high-throughput format. Because of these attributes, the method could find utility in drug discovery applications such as characterizing the effect of pharmacological inhibitors on Wnt signaling without the need for sophisticated biophysical instrumentation.
Asunto(s)
Receptores Frizzled , Vía de Señalización Wnt , Proteína Wnt3A , Animales , Ensayo de Inmunoadsorción Enzimática , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Humanos , Unión Proteica , Proteína Wnt3A/química , Proteína Wnt3A/metabolismoRESUMEN
Wnt signaling regulates physiological processes ranging from cell differentiation to bone formation. Dysregulation of Wnt signaling is linked to several human ailments, including colorectal, pancreatic, and breast cancers. As such, modulation of this pathway has been an attractive strategy for therapeutic development of anticancer agents. Since the discovery of Wnt proteins more than 35 years ago, research efforts continue to focus on understanding the biochemistry of their molecular interactions and their biological functions. Wnt is a secreted glycoprotein covalently modified with a cis-unsaturated fatty acyl group at a conserved serine residue, and this modification is required for Wnt secretion and activity. To initiate signaling, Wnt proteins bind to cell-surface Frizzled (FZD) receptors, but the molecular basis for recognition of Wnt's fatty acyl moiety by the extracellular cysteine-rich domain of FZD has become clear only very recently. Here, we review the most recent developments in the field, focusing on structural and biochemical studies of the FZD receptor family and highlighting new insights into their molecular arrangement and mode of regulation by cis-unsaturated fatty acids. Additionally, we examine how other lipid-binding proteins recognize fatty acyl chains on Wnt proteins in the regulation of Wnt secretion and activities. Altogether, this perspective expands our understanding of fatty acid-protein interactions in the FZD system and provides a basis for guiding future research in the field.
Asunto(s)
Ácidos Grasos/metabolismo , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Familia de Multigenes , Animales , Sitios de Unión , Cristalografía por Rayos X , Ácidos Grasos/química , Receptores Frizzled/genética , Humanos , Modelos Moleculares , Transducción de SeñalRESUMEN
Sec14, the major yeast phosphatidylcholine (PC)/phosphatidylinositol (PI) transfer protein (PITP), coordinates PC and PI metabolism to facilitate an appropriate and essential lipid signaling environment for membrane trafficking from trans-Golgi membranes. The Sec14 PI/PC exchange cycle is essential for its essential biological activity, but fundamental aspects of how this PITP executes its lipid transfer cycle remain unknown. To address some of these outstanding issues, we applied time-resolved small-angle neutron scattering for the determination of protein-mediated intervesicular movement of deuterated and hydrogenated phospholipids in vitro. Quantitative analysis by small-angle neutron scattering revealed that Sec14 PI- and PC-exchange activities were sensitive to both the lipid composition and curvature of membranes. Moreover, we report that these two parameters regulate lipid exchange activity via distinct mechanisms. Increased membrane curvature promoted both membrane binding and lipid exchange properties of Sec14, indicating that this PITP preferentially acts on the membrane site with a convexly curved face. This biophysical property likely constitutes part of a mechanism by which spatial specificity of Sec14 function is determined in cells. Finally, wild-type Sec14, but not a mixture of Sec14 proteins specifically deficient in either PC- or PI-binding activity, was able to effect a net transfer of PI or PC down opposing concentration gradients in vitro.
Asunto(s)
Fosfatidilcolinas/metabolismo , Fosfatidilinositoles/metabolismo , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Saccharomyces cerevisiae/química , Difracción de Neutrones , Fosfatidilcolinas/química , Fosfatidilinositoles/química , Proteínas de Transferencia de Fosfolípidos/metabolismo , Unión Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Dispersión del Ángulo Pequeño , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismoRESUMEN
The version of this article originally published contained older versions of the Life Sciences Reporting Summary and the Supplementary Text and Figures. The error has been corrected in the HTML and PDF versions of the article.
RESUMEN
Regeneration of the adult intestinal epithelium is mediated by a pool of cycling stem cells, which are located at the base of the crypt, that express leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5). The Frizzled (FZD) 7 receptor (FZD7) is enriched in LGR5+ intestinal stem cells and plays a critical role in their self-renewal. Yet, drug discovery approaches and structural bases for targeting specific FZD isoforms remain poorly defined. FZD proteins interact with Wnt signaling proteins via, in part, a lipid-binding groove on the extracellular cysteine-rich domain (CRD) of the FZD receptor. Here we report the identification of a potent peptide that selectively binds to the FZD7 CRD at a previously uncharacterized site and alters the conformation of the CRD and the architecture of its lipid-binding groove. Treatment with the FZD7-binding peptide impaired Wnt signaling in cultured cells and stem cell function in intestinal organoids. Together, our data illustrate that targeting the lipid-binding groove holds promise as an approach for achieving isoform-selective FZD receptor inhibition.
Asunto(s)
Receptores Frizzled/antagonistas & inhibidores , Receptores Frizzled/metabolismo , Intestinos/efectos de los fármacos , Células Madre/efectos de los fármacos , Animales , Sitios de Unión , Células CHO , Membrana Celular/metabolismo , Cricetulus , Cristalografía por Rayos X , Descubrimiento de Drogas , Femenino , Citometría de Flujo , Células HEK293 , Humanos , Intestinos/citología , Lípidos/química , Ratones , Ratones Endogámicos C57BL , Péptidos/química , Unión Proteica , Multimerización de Proteína , Regeneración , Análisis de Secuencia de ARN , Transducción de Señal/efectos de los fármacos , Células Madre/patología , Resonancia por Plasmón de Superficie , Vía de Señalización WntRESUMEN
Frizzled (FZD) receptors mediate Wnt signaling in diverse processes ranging from bone growth to stem cell activity. Moreover, high FZD receptor expression at the cell surface contributes to overactive Wnt signaling in subsets of pancreatic, ovarian, gastric, and colorectal tumors. Despite the progress in biochemical understanding of Wnt-FZD receptor interactions, the molecular basis for recognition of Wnt cis-unsaturated fatty acyl groups by the cysteine-rich domain (CRD) of FZD receptors remains elusive. Here, we determined a crystal structure of human FZD7 CRD unexpectedly bound to a 24-carbon fatty acid. We also report a crystal structure of human FZD5 CRD bound to C16:1 cis-Δ9 unsaturated fatty acid. Both structures reveal a dimeric arrangement of the CRD. The lipid-binding groove exhibits flexibility and spans both monomers, adopting a U-shaped geometry that accommodates the fatty acid. Re-evaluation of the published mouse FZD8 CRD structure reveals that it also shares the same architecture as FZD5 and FZD7 CRDs. Our results define a common molecular mechanism for recognition of the cis-unsaturated fatty acyl group, a necessary posttranslational modification of Wnts, by multiple FZD receptors. The fatty acid bridges two CRD monomers, implying that Wnt binding mediates FZD receptor dimerization. Our data uncover possibilities for the arrangement of Wnt-FZD CRD complexes and shed structural insights that could aide in the identification of pharmacological strategies to modulate FZD receptor function.
Asunto(s)
Cisteína/química , Ácidos Grasos Insaturados/química , Receptores Frizzled/química , Proteínas Wnt/química , beta Catenina/química , Cristalografía por Rayos X , Cisteína/metabolismo , Ácidos Grasos Insaturados/metabolismo , Receptores Frizzled/metabolismo , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
Wnt proteins are critical regulators of signaling networks during embryonic development and in adult tissue homeostasis. The generation of active Wnt proteins requires their regulated secretion into the extracellular space. Once secreted, Wnts signal through the cell surface via receptor binding on Wnt-receiving cells, a mechanism that is prevalent in stem cell and cancer biology. Important to both Wnt secretion and receptor recognition is their post-translational fatty acylation. In this Perspective, we highlight progress in elucidating the biochemistry of Wnt fatty acylation and provide a molecular view on the enzymology of substrate recognition and catalysis, with a focus on the Wnt O-acyltransferase porcupine. Special emphasis is given to Wnt fatty acid biosynthesis, Wnt-porcupine interactions, clinical mutations of porcupine and emerging therapeutics for perturbing Wnt fatty acylation in cancer. Finally, we discuss models for the functional role of the unsaturated fatty acyl chain in mediating lipid-protein interactions and in Wnt trafficking.
Asunto(s)
Acetiltransferasas/metabolismo , Ácidos Grasos Insaturados/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Acilación , Secuencia de Aminoácidos , Animales , Sitios de Unión , Membrana Celular/enzimología , Secuencia Conservada , Ácidos Grasos Insaturados/química , Humanos , Modelos Moleculares , Estructura Molecular , Mutación , Isoformas de Proteínas/genética , Alineación de Secuencia , Proteínas Wnt/química , Proteínas Wnt/genéticaRESUMEN
The physiological functions of phosphatidylinositol (PtdIns)-transfer proteins (PITPs)/phosphatidylcholine (PtdCho)-transfer proteins are poorly characterized, even though these proteins are conserved throughout the eukaryotic kingdom. Much of the progress in elucidating PITP functions has come from exploitation of genetically tractable model organisms, but the mechanisms for how PITPs execute their biological activities remain unclear. Structural and molecular dynamics approaches are filling in the details for how these proteins actually work as molecules. In the present paper, we discuss our recent work with Sec14-like PITPs and describe how PITPs integrate diverse territories of the lipid metabolome with phosphoinositide signalling.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Modelos Biológicos , Fosfatidilcolinas/metabolismo , Fosfatidilinositoles/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sistemas de Mensajero Secundario , Animales , Sitios de Unión , Transporte Biológico , Proteínas de Ciclo Celular/química , Proteínas Cromosómicas no Histona/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Fosfatidilcolinas/química , Fosfatidilinositoles/química , Proteínas de Transferencia de Fosfolípidos/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.