RESUMEN
Kidney cancer, one of the ten most prevalent malignancies in the world, has exhibited increased incidence over the last decade. The most common subtype is "clear cell" renal cell carcinoma (ccRCC), which features consistent metabolic abnormalities, such as highly elevated glycogen and lipid deposition. By integrating metabolomics, genomic, and transcriptomic data, we determined that enzymes in multiple metabolic pathways are universally depleted in human ccRCC tumors, which are otherwise genetically heterogeneous. Notably, the expression of key urea cycle enzymes, including arginase 2 (ARG2) and argininosuccinate synthase 1 (ASS1), is strongly repressed in ccRCC. Reduced ARG2 activity promotes ccRCC tumor growth through at least two distinct mechanisms: conserving the critical biosynthetic cofactor pyridoxal phosphate and avoiding toxic polyamine accumulation. Pharmacological approaches to restore urea cycle enzyme expression would greatly expand treatment strategies for ccRCC patients, where current therapies only benefit a subset of those afflicted with renal cancer.
Asunto(s)
Arginasa/metabolismo , Carcinoma de Células Renales/patología , Neoplasias Renales/patología , Poliaminas/metabolismo , Animales , Arginasa/genética , Argininosuccinato Sintasa/metabolismo , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/enzimología , Línea Celular Tumoral , Perfilación de la Expresión Génica , Xenoinjertos , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/enzimología , Ratones , Ratones Desnudos , Fosfato de Piridoxal/metabolismo , Urea/metabolismoRESUMEN
Protein prenylation is a post-translational modification that is responsible for membrane association and protein-protein interactions. The oncogenic protein Ras, which is prenylated, has been the subject of intense study in the past 20 years as a therapeutic target. Several studies have shown a correlation between neurodegenerative diseases including Alzheimer's disease and Parkinson's disease and protein prenylation. Here, a method for imaging and quantification of the prenylome using microscopy and flow cytometry is described. We show that metabolically incorporating an alkyne isoprenoid into mammalian cells, followed by a Cu(I)-catalyzed alkyne azide cycloaddition reaction to a fluorophore, allows for detection of prenylated proteins in several cell lines and that different cell types vary significantly in their levels of prenylated proteins. The addition of a prenyltransferase inhibitor or the precursors to the native isoprenoid substrates lowers the levels of labeled prenylated proteins. Finally, we demonstrate that there is a significantly higher (22%) level of prenylated proteins in a cellular model of compromised autophagy as compared to normal cells, supporting the hypothesis of a potential involvement of protein prenylation in abrogated autophagy. These results highlight the utility of total prenylome labeling for studies on the role of protein prenylation in various diseases including aging-related disorders.
Asunto(s)
Alquinos/química , Prenilación de Proteína , Terpenos/química , Autofagia , Citometría de Flujo , Células HeLa , HumanosRESUMEN
In soft tissue sarcomas (STS), low intratumoural O2 (hypoxia) is a poor prognostic indicator. HIF-1α mediates key transcriptional responses to hypoxia, and promotes STS metastasis; however, the role of the related HIF-2α protein is unknown. Surprisingly, here we show that HIF-2α inhibits high-grade STS cell growth in vivo, as loss of HIF-2α promotes sarcoma proliferation and increases calcium and mTORC1 signalling in undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma. We find that most human STS have lower levels of EPAS1 (the gene encoding HIF-2α) expression relative to normal tissue. Many cancers, including STS, contain altered epigenetics, and our findings define an epigenetic mechanism whereby EPAS1 is silenced during sarcoma progression. The clinically approved HDAC inhibitor Vorinostat specifically increases HIF-2α, but not HIF-1α, accumulation in multiple STS subtypes. Vorinostat inhibits STS tumour growth, an effect ameliorated by HIF-2α deletion, implicating HIF-2α as a biomarker for Vorinostat efficacy in STS.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proliferación Celular/genética , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Hipoxia/genética , Liposarcoma/genética , Complejos Multiproteicos/metabolismo , Sarcoma/genética , Serina-Treonina Quinasas TOR/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Señalización del Calcio/genética , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Células HEK293 , Miembro Posterior , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Immunoblotting , Inmunohistoquímica , Técnicas In Vitro , Liposarcoma/diagnóstico por imagen , Liposarcoma/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Trasplante de Neoplasias , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcoma/diagnóstico por imagen , Sarcoma/metabolismo , Transducción de Señal/genética , Tomografía Computarizada por Rayos X , VorinostatRESUMEN
UNLABELLED: Two hallmarks of clear-cell renal cell carcinoma (ccRCC) are constitutive hypoxia-inducible factor (HIF) signaling and abundant intracellular lipid droplets (LD). However, regulation of lipid storage and its role in ccRCC are incompletely understood. Transcriptional profiling of primary ccRCC samples revealed that expression of the LD coat protein gene PLIN2 was elevated in tumors and correlated with HIF2α, but not HIF1α, activation. HIF2α-dependent PLIN2 expression promoted lipid storage, proliferation, and viability in xenograft tumors. Mechanistically, lipid storage maintained integrity of the endoplasmic reticulum (ER), which is functionally and physically associated with LDs. Specifically, PLIN2-dependent lipid storage suppressed cytotoxic ER stress responses that otherwise result from elevated protein synthetic activity characteristic of ccRCC cells. Thus, in addition to promoting ccRCC proliferation and anabolic metabolism, HIF2α modulates lipid storage to sustain ER homeostasis, particularly under conditions of nutrient and oxygen limitation, thereby promoting tumor cell survival. SIGNIFICANCE: We demonstrate that HIF2α promotes lipid storage, ER homeostasis, and cell viability in ccRCC via upregulation of the LD coat protein PLIN2, revealing a novel function for the well-documented "clear-cell" phenotype and identifying ER stress as a targetable vulnerability created by HIF2α/PLIN2 suppression in this common renal malignancy.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Células Renales/metabolismo , Retículo Endoplásmico/metabolismo , Homeostasis , Neoplasias Renales/metabolismo , Metabolismo de los Lípidos , Animales , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Estrés del Retículo Endoplásmico , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Neoplasias Renales/genética , Neoplasias Renales/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Oncogenes , Perilipina-2 , Biosíntesis de Proteínas , Carga Tumoral , Respuesta de Proteína DesplegadaRESUMEN
Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is characterized by elevated glycogen levels and fat deposition. These consistent metabolic alterations are associated with normoxic stabilization of hypoxia-inducible factors (HIFs) secondary to von Hippel-Lindau (VHL) mutations that occur in over 90% of ccRCC tumours. However, kidney-specific VHL deletion in mice fails to elicit ccRCC-specific metabolic phenotypes and tumour formation, suggesting that additional mechanisms are essential. Recent large-scale sequencing analyses revealed the loss of several chromatin remodelling enzymes in a subset of ccRCC (these included polybromo-1, SET domain containing 2 and BRCA1-associated protein-1, among others), indicating that epigenetic perturbations are probably important contributors to the natural history of this disease. Here we used an integrative approach comprising pan-metabolomic profiling and metabolic gene set analysis and determined that the gluconeogenic enzyme fructose-1,6-bisphosphatase 1 (FBP1) is uniformly depleted in over six hundred ccRCC tumours examined. Notably, the human FBP1 locus resides on chromosome 9q22, the loss of which is associated with poor prognosis for ccRCC patients. Our data further indicate that FBP1 inhibits ccRCC progression through two distinct mechanisms. First, FBP1 antagonizes glycolytic flux in renal tubular epithelial cells, the presumptive ccRCC cell of origin, thereby inhibiting a potential Warburg effect. Second, in pVHL (the protein encoded by the VHL gene)-deficient ccRCC cells, FBP1 restrains cell proliferation, glycolysis and the pentose phosphate pathway in a catalytic-activity-independent manner, by inhibiting nuclear HIF function via direct interaction with the HIF inhibitory domain. This unique dual function of the FBP1 protein explains its ubiquitous loss in ccRCC, distinguishing FBP1 from previously identified tumour suppressors that are not consistently mutated in all tumours.
Asunto(s)
Carcinoma de Células Renales/enzimología , Fructosa-Bifosfatasa/metabolismo , Neoplasias Renales/enzimología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/fisiopatología , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Fructosa-Bifosfatasa/química , Fructosa-Bifosfatasa/genética , Glucólisis , Humanos , Neoplasias Renales/genética , Neoplasias Renales/fisiopatología , Modelos Moleculares , NADP/metabolismo , Estructura Terciaria de Proteína , PorcinosRESUMEN
Protein prenylation is a post-translational modification required for proper cellular localization and activity of many important eukaryotic proteins. Farnesyltransferase inhibitors (FTIs) have been explored extensively for their antitumor activity. To assist in identifying potentially new and more useful markers for therapeutic applications, we developed a strategy that uses a combination of metabolic labeling and 2D DIGE (differential gel electrophoresis) to discover new prenylated proteins whose cellular levels are influenced by FTIs. In this approach, metabolic labeling of prenylated proteins was first carried out with an alkyne-modified isoprenoid analog, C15Alk, in the presence or absence of the FTI L-744,832. The resulting alkyne-tagged proteins were then labeled with Cy3-N3 and Cy5-N3 and subjected to 2D-DIGE. Multiple spots having altered levels of labeling in presence of the FTI were observed. Mass spectrometric analysis of some of the differentially labeled spots identified several known prenylated proteins, along with HisRS, PACN-3, GNAI-1 and GNAI-2, which are not known to be prenylated. In vitro farnesylation of a C-terminal peptide sequence derived from GNAI-1 and GNAI-2 produced a farnesylated product, suggesting GNAI-1 and GNAI-2 are potential novel farnesylated proteins. These results suggest that this new strategy could be useful for the identification of prenylated proteins whose level of post-translational modification has been modulated by the presence of an FTI. Additionally, this approach, which decreases sample complexity and thereby facilitates analysis, should be applicable to studies of other post-translational modifications as well.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Prenilación de Proteína/efectos de los fármacos , Coloración y Etiquetado , Electroforesis Bidimensional Diferencial en Gel/métodos , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Fraccionamiento Químico , Inhibidores Enzimáticos/química , Farnesiltransferasa/metabolismo , Subunidad alfa de la Proteína de Unión al GTP Gi2/química , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Células HeLa , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Octoxinol , Polietilenglicoles/farmacologíaRESUMEN
Protein prenylation involves the addition of a farnesyl (C15) or geranylgeranyl (C20) isoprenoid moiety onto the C-terminus of approximately 2 % of all mammalian proteins. This hydrophobic modification serves to direct membrane association of the protein. Due to the finding that the oncogenic protein Ras is naturally prenylated, several researchers have developed inhibitors of the prenyltransferase enzymes as cancer therapeutics. Despite numerous studies on the enzymology of prenylation in vitro, many questions remain about the process of prenylation in living cells. Using a combination of flow cytometry and confocal microscopy, we have shown that synthetic fluorescently labeled prenylated peptides enter a variety of different cell types. Additionally, using capillary electrophoresis we have shown that these peptides can be detected in minute quantities from lysates of cells treated with these peptides. This method will allow for further study of the enzymology of protein prenylation in living cells.
Asunto(s)
Imagen Molecular/métodos , Péptidos , Prenilación de Proteína , Animales , Línea Celular Transformada , Separación Celular , Cromatografía Capilar Electrocinética Micelar , Citometría de Flujo , Colorantes Fluorescentes/química , Células HeLa , Humanos , Ratones , Microscopía Confocal , Neuronas/citología , Péptidos/química , RatasRESUMEN
Stem cells exert precise regulation to maintain a balance of self-renewal and differentiation programs to sustain tissue homeostasis throughout the life of an organism. Recent evidence suggests that this regulation is modulated, in part, via metabolic changes and modifications of nutrient-sensing pathways such as mTOR and AMPK. It is becoming increasingly clear that stem cells inhibit oxidative phosphorylation in favor of aerobic glycolysis for energy production. Recent progress has detailed the molecular mechanisms of this metabolic phenotype and has offered insight into new metabolic pathways that may be involved in stem cell homeostasis.
Asunto(s)
Metabolismo Energético , Transducción de Señal , Células Madre/metabolismo , Adaptación Fisiológica , Animales , Diferenciación Celular , Proliferación Celular , Glucólisis , Homeostasis , Humanos , Fosforilación Oxidativa , Nicho de Células MadreRESUMEN
The posttranslational modification of protein prenylation is a covalent lipid modification on the C-terminus of substrate proteins that serves to enhance membrane affinity. Oncogenic proteins such as Ras have this modification and significant effort has been placed into developing inhibitors of the prenyltransferase enzymes for clinical therapy. In addition to cancer therapy, prenyltransferase inhibitors have begun to find important therapeutic uses in other diseases, including progeria, hepatitis C and D, parasitic infections, and other maladies. This review will trace the evolution of prenyltransferase inhibitors from their initial use as cancer therapeutics to their expanded applications for other diseases.
RESUMEN
The creation of caged molecules involves the attachment of protecting groups to biologically active compounds such as ligands, substrates and drugs that can be removed under specific conditions. Photoremovable caging groups are the most common due to their ability to be removed with high spatial and temporal resolution. Here, the synthesis and photochemistry of a caged inhibitor of protein farnesyltransferase is described. The inhibitor, FTI, was caged by alkylation of a critical thiol group with a bromohydroxycoumarin (Bhc) moiety. While Bhc is well established as a protecting group for carboxylates and phosphates, it has not been extensively used to cage sulfhydryl groups. The resulting caged molecule, Bhc-FTI, can be photolyzed with UV light to release the inhibitor that prevents Ras farnesylation, Ras membrane localization and downstream signaling. Finally, it is shown that Bhc-FTI can be uncaged by two-photon excitation to produce FTI at levels sufficient to inhibit Ras localization and alter cell morphology. Given the widespread involvement of Ras proteins in signal transduction pathways, this caged inhibitor should be useful in a plethora of studies.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Farnesiltransferasa/antagonistas & inhibidores , Fotones , Proteínas ras/antagonistas & inhibidores , Animales , Línea Celular , Cumarinas/química , Perros , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/química , Farnesiltransferasa/metabolismo , Humanos , Procesos Fotoquímicos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Espectrometría de Fluorescencia , Proteínas ras/metabolismoRESUMEN
Protein prenylation involves the addition of either a farnesyl (C(15)) or geranylgeranyl (C(20)) isoprenoid moiety onto the C-terminus of many proteins. This natural modification serves to direct a protein to the plasma membrane of the cell. A recently discovered application of prenylated peptides is that they have inherent cell-penetrating ability, and are hence termed cell penetrating prenylated peptides. These peptides are able to efficiently cross the cell membrane in an ATP independent, non-endocytotic manner and it was found that the sequence of the peptide does not affect uptake, so long as the geranylgeranyl group is still present [Wollack, J. W.; Zeliadt, N. A.; Mullen, D. G.; Amundson, G.; Geier, S.; Falkum, S.; Wattenberg, E. V.; Barany, G.; Distefano, M. D. Multifunctional Prenylated Peptides for Live Cell Analysis. J. Am. Chem. Soc.2009, 131, 7293-7303]. The present study investigates the effect of removing the fluorophore from the peptides and investigating the uptake by confocal microscopy and flow cytometry. Our results show that the fluorophore is not necessary for uptake of these peptides. This information is significant because it indicates that the prenyl group is the major determinant in allowing these peptides to enter cells; the hydrophobic fluorophore has little effect. Moreover, these studies demonstrate the utility of the Cu-catalyzed click reaction for monitoring the entry of nonfluorescent peptides into cells.
Asunto(s)
Colorantes Fluorescentes/química , Péptidos/química , Prenilación de Proteína , Citometría de Flujo , Células HeLa , Humanos , Microscopía ConfocalRESUMEN
Protein prenyltransferases catalyze the attachment of C15 (farnesyl) and C20 (geranylgeranyl) groups to proteins at specific sequences localized at or near the C-termini of specific proteins. Determination of the specific protein prenyltransferase substrates affected by the inhibition of these enzymes is critical for enhancing knowledge of the mechanism of such potential drugs. Here, we investigate the utility of alkyne-containing isoprenoid analogs for chemical proteomics experiments by showing that these compounds readily penetrate mammalian cells in culture and become incorporated into proteins that are normally prenylated. Derivatization via Cu(I) catalyzed click reaction with a fluorescent azide reagent allows the proteins to be visualized and their relative levels to be analyzed. Simultaneous treatment of cells with these probes and inhibitors of prenylation reveals decreases in the levels of some but not all of the labeled proteins. Two-dimensional electrophoretic separation of these labeled proteins followed by mass spectrometric analysis allowed several labeled proteins to be unambiguously identified. Docking experiments and density functional theory calculations suggest that the substrate specificity of protein farnesyl transferase may vary depending on whether azide- or alkyne-based isoprenoid analogs is employed. These results demonstrate the utility of alkyne-containing analogs for chemical proteomic applications.
Asunto(s)
Alquinos/química , Azidas/química , Prenilación de Proteína , Proteómica/métodos , Terpenos/química , Animales , Biomarcadores/química , Dominio Catalítico , Línea Celular , Electroforesis en Gel Bidimensional , Células HeLa , Humanos , Teoría Cuántica , Especificidad por SustratoRESUMEN
Protein prenylation is a posttranslational modification that is present in a large number of proteins; it has been proposed to be responsible for membrane association and protein-protein interactions, which contribute to its role in signal transduction pathways. Research has been aimed at inhibiting prenylation with farnesyltransferase inhibitors based on the finding that the farnesylated protein Ras is implicated in 30% of human cancers. Despite numerous studies on the enzymology of prenylation in vitro, many questions remain about the process of prenylation as it occurs in living cells. Here we describe the preparation of a series of farnesylated peptides that contain sequences recognized by protein farnesyltransferase. Using a combination of flow cytometry and confocal microscopy, we show that these peptides enter a variety of different cell types. A related peptide where the farnesyl group has been replaced by a disulfide-linked decyl group is also shown to be able to efficiently enter cells. These results highlight the applicability of these peptides as a platform for further study of protein prenylation and subsequent processing in live cells.
Asunto(s)
Péptidos/química , Prenilación de Proteína , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Farnesiltransferasa/metabolismo , Citometría de Flujo , Colorantes Fluorescentes/química , Humanos , Ratones , Microscopía Confocal , Péptidos/síntesis química , Péptidos/farmacologíaRESUMEN
The similarity ensemble approach (SEA) relates proteins based on the set-wise chemical similarity among their ligands. It can be used to rapidly search large compound databases and to build cross-target similarity maps. The emerging maps relate targets in ways that reveal relationships one might not recognize based on sequence or structural similarities alone. SEA has previously revealed cross talk between drugs acting primarily on G-protein coupled receptors (GPCRs). Here we used SEA to look for potential off-target inhibition of the enzyme protein farnesyltransferase (PFTase) by commercially available drugs. The inhibition of PFTase has profound consequences for oncogenesis, as well as a number of other diseases. In the present study, two commercial drugs, Loratadine and Miconazole, were identified as potential ligands for PFTase and subsequently confirmed as such experimentally. These results point toward the applicability of SEA for the prediction of not only GPCR-GPCR drug cross talk but also GPCR-enzyme and enzyme-enzyme drug cross talk.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Loratadina/farmacología , Miconazol/farmacología , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Farnesiltransferasa/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Antagonistas de los Receptores Histamínicos H1 no Sedantes/química , Antagonistas de los Receptores Histamínicos H1 no Sedantes/metabolismo , Antagonistas de los Receptores Histamínicos H1 no Sedantes/farmacología , Ligandos , Loratadina/química , Loratadina/metabolismo , Miconazol/química , Miconazol/metabolismo , Microscopía Confocal , Estructura Molecular , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , Mapeo de Interacción de Proteínas/métodos , Prenilación de Proteína/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tecnología Farmacéutica/métodos , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Cell penetrating peptides are useful delivery tools for introducing molecules of interest into cells. A new class of cell penetrating molecules has been recently reported-cell penetrating, prenylated peptides. In this study a series of such peptides was synthesized to examine the relationship between peptide sequence and level of peptide internalization and to probe their mechanism of internalization. This study revealed that prenylated peptides internalize via a non-endocytotic pathway regardless of sequence. Sequence length and identity was found to play a role in peptide uptake but prenylated sequences as short as two amino acids were found to exhibit significant cell penetrating properties.