RESUMEN
Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.
Asunto(s)
Proteínas Hedgehog/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Cromatografía de Afinidad , Proteómica , Transducción de Señal , Espectrometría de Masas en TándemRESUMEN
Targeted degradation of proteins through the ubiquitin-proteasome system (UPS) via the activities of E3 ubiquitin ligases regulates diverse cellular processes, and misregulation of these enzymes contributes to the pathogenesis of human diseases. One of the challenges facing the UPS field is to delineate the complete cohort of substrates for a particular E3 ligase. Advances in mass spectrometry and the development of antibodies recognizing the Lys-ϵ-Gly-Gly (diGly) remnant from ubiquitinated proteins following trypsinolysis have provided a tool to address this question. We implemented an inducible loss of function approach in combination with quantitative diGly proteomics to find novel substrates of HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase), an E3 ligase implicated in cancer and intellectual disabilities. diGly proteomics results led to the identification of DNA damage-inducible transcript 4 (DDIT4) as a putative HUWE1 substrate. Cell-based assays demonstrated that HUWE1 interacts with and regulates ubiquitination and stability of DDIT4. Together these data suggest a model in which HUWE1 mediates DDIT4 proteasomal degradation. Our results demonstrate proof of concept that inducible knockdown of an E3 ligase in combination with diGly proteomics provides a potentially advantageous method for identifying novel E3 substrates that may help to identify candidates for therapeutic modulation in the UPS.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Oligopéptidos/química , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisina/química , Espectrometría de Masas , Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteómica , Interferencia de ARN , Proteínas Supresoras de Tumor , Ubiquitina/química , Ubiquitina Tiolesterasa/metabolismo , Peptidasa Específica de Ubiquitina 7RESUMEN
Aggregates of hyperphosphorylated tau protein are a pathological hallmark of more than 20 distinct neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, and frontotemporal dementia. While the exact mechanism of tau aggregation is unknown, the accumulation of aggregates correlates with disease progression. Here we report a genome-wide CRISPR screen to identify modulators of endogenous tau protein for the first time. Primary screens performed in SH-SY5Y cells, identified positive and negative regulators of tau protein levels. Hit validation of the top 43 candidate genes was performed using Ngn2-induced human cortical excitatory neurons. Using this approach, genes and pathways involved in modulation of endogenous tau levels were identified, including chromatin modifying enzymes, neddylation and ubiquitin pathway members, and components of the mTOR pathway. TSC1, a critical component of the mTOR pathway, was further validated in vivo, demonstrating the relevance of this screening strategy. These findings may have implications for treating neurodegenerative diseases in the future.
Asunto(s)
Redes y Vías Metabólicas/genética , Neuronas/metabolismo , Proteínas tau/metabolismo , Animales , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Línea Celular Tumoral , Edición Génica , Genes/genética , Genes/fisiología , Pruebas Genéticas/métodos , Estudio de Asociación del Genoma Completo , Humanos , Ratones , Neuroblastoma/metabolismo , Ratas , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Aberrant activation of the Hedgehog (Hh) pathway can drive tumorigenesis. To investigate the mechanism by which glioma-associated oncogene family zinc finger-1 (GLI1), a crucial effector of Hh signaling, regulates Hh pathway activation, we searched for GLI1-interacting proteins. We report that the chromatin remodeling protein SNF5 (encoded by SMARCB1, hereafter called SNF5), which is inactivated in human malignant rhabdoid tumors (MRTs), interacts with GLI1. We show that Snf5 localizes to Gli1-regulated promoters and that loss of Snf5 leads to activation of the Hh-Gli pathway. Conversely, re-expression of SNF5 in MRT cells represses GLI1. Consistent with this, we show the presence of a Hh-Gli-activated gene expression profile in primary MRTs and show that GLI1 drives the growth of SNF5-deficient MRT cells in vitro and in vivo. Therefore, our studies reveal that SNF5 is a key mediator of Hh signaling and that aberrant activation of GLI1 is a previously undescribed targetable mechanism contributing to the growth of MRT cells.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Tumor Rabdoide/genética , Transducción de Señal/genética , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/genética , Cartilla de ADN/genética , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Humanos , Immunoblotting , Hibridación in Situ , Espectrometría de Masas , Ratones , Análisis por Micromatrices , Proteína SMARCB1 , Factores de Transcripción/genética , Proteína con Dedos de Zinc GLI1RESUMEN
Deubiquitinating enzymes regulate essential cellular processes, and their dysregulation is implicated in multiple disease states. Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) has garnered attention for its links with Parkinson's disease and cancer; however, the mechanism of action of this enzyme in cells remains poorly understood. In order to advance our understanding of UCH-L1 function, we have been developing small molecule modulators of the enzyme for use as tools to probe its role in cells. In support of these efforts, an investigation of the mechanism of UCH-L1 catalysis was previously reported. Here, we extend this mechanistic evaluation and examine substrate recognition by UCH-L1. We developed a panel of ubiquitin fusions to test the contribution of specific residues of ubiquitin to binding and catalysis by the enzyme, and determined the activation parameters of selected variants to gain additional mechanistic insight. Ubiquitin side chains critical for establishing the Michaelis complex and enabling catalysis were identified, and features of this complex that differ between UCH-L1 and a homologue, UCH-L3, were revealed. These data provide dramatic examples of differences in substrate specificity between these enzymes. The implications of our experiments with UCH-L1 for selective inhibitor design and the relationship to disease are discussed.
Asunto(s)
Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/metabolismo , Sustitución de Aminoácidos , Catálisis , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Humanos , Cinética , Modelos Moleculares , Enfermedad de Parkinson/enzimología , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Termodinámica , Ubiquitina/metabolismoRESUMEN
The ubiquitin C-terminal hydrolase UCH-L1 (PGP9.5) comprises >1% of total brain protein but is almost absent from other tissues [Wilkinson, K. D., et al. (1989) Science 246, 670-673]. Mutations in the UCH-L1 gene have been reported to be linked to susceptibility to and protection from Parkinson's disease [Leroy, E., et al. (1998) Nature 395, 451-452; Maraganore, D. M., et al. (1999) Neurology 53, 1858-1860]. Abnormal overexpression of UCH-L1 has been shown to correlate with several forms of cancer [Hibi, K., et al. (1998) Cancer Res. 58, 5690-5694]. Because the amino acid sequence of UCH-L1 is similar to that of other ubiquitin C-terminal hydrolases, including the ubiquitously expressed UCH-L3, which appear to be unconnected to neurodegenerative disease, the structure of UCH-L1 and the effects of disease associated mutations on the structure and function are of considerable importance. We have determined the three-dimensional structure of human UCH-L1 at 2.4-A resolution by x-ray crystallography. The overall fold resembles that of other ubiquitin hydrolases, including UCH-L3, but there are a number of significant differences. In particular, the geometry of the catalytic residues in the active site of UCH-L1 is distorted in such a way that the hydrolytic activity would appear to be impossible without substrate induced conformational rearrangements.
Asunto(s)
Enfermedad de Parkinson/enzimología , Ubiquitina Tiolesterasa/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
Glycoproteins are essential for cellular communication and are the most rapidly growing class of therapeutic agents. Chemical modification of glycoproteins has been employed to improve their in vivo efficacy or to label them for detection. Methods for the controlled derivatization of glycoproteins are presently limited by the repertoire of natural amino acid side chain and carbohydrate functionalities. Here, we use metabolic oligosaccharide engineering to introduce a bioorthogonal functional group, the azide, into cellular and recombinant glycoproteins for subsequent chemical elaboration via Staudinger ligation. As most therapeutic glycoproteins are sialylated and require this saccharide for optimal pharmacokinetics, we targeted sialic acid as a host for azides using N-azidoacetylmannosamine (ManNAz) as a biosynthetic precursor. Metabolic conversion of ManNAz to N-azidoacetylsialic acid (SiaNAz) within membrane-bound and secreted glycoproteins was quantified in a variety of cell types. SiaNAz was found to comprise between 4% and 41% of total sialosides, depending on the system. Metabolic labeling of recombinant interferon-beta and GlyCAM-Ig was achieved, demonstrating the utility of the method for functionalizing N-linked and O-linked glycoproteins of therapeutic interest. More generally, the generation of recombinant glycoproteins containing chemical handles within their glycans provides a means for studying their behavior and for improving their in vivo efficacy.
Asunto(s)
Glicoproteínas/metabolismo , Proteínas Recombinantes/metabolismo , Animales , Azidas/química , Azidas/metabolismo , Azidas/farmacología , Línea Celular , Chlorocebus aethiops , Cromatografía Líquida de Alta Presión , Cricetinae , Expresión Génica , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Espectrometría de Masas , Estructura Molecular , Ácido N-Acetilneuramínico/metabolismo , Oligosacáridos/química , Oligosacáridos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Sialic acid is a major determinant of carbohydrate-receptor interactions in many systems pertinent to human health and disease. N-Acetylmannosamine (ManNAc) is the first committed intermediate in the sialic acid biosynthetic pathway; thus, the mechanisms that control intracellular ManNAc levels are important regulators of sialic acid production. UDP-GlcNAc 2-epimerase and GlcNAc 2-epimerase are two enzymes capable of generating ManNAc from UDP-GlcNAc and GlcNAc, respectively. Whereas the former enzyme has been shown to direct metabolic flux toward sialic acid in vivo, the function of the latter enzyme is unclear. Here we study the effects of GlcNAc 2-epimerase expression on sialic acid production in cells. A key tool we developed for this study is a cell-permeable, small molecule inhibitor of GlcNAc 2-epimerase designed based on mechanistic principles. Our results indicate that, unlike UDP-GlcNAc 2-epimerase, which promotes biosynthesis of sialic acid, GlcNAc 2-epimerase can serve a catabolic role, diverting metabolic flux away from the sialic acid pathway.
Asunto(s)
Carbohidrato Epimerasas/metabolismo , Proteínas Portadoras/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Secuencia de Bases , Carbohidrato Epimerasas/antagonistas & inhibidores , Carbohidrato Epimerasas/genética , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Cartilla de ADN , Inhibidores Enzimáticos/farmacología , Humanos , Células Jurkat , Resonancia Magnética Nuclear Biomolecular , ARN Mensajero/genética , Especificidad por SustratoRESUMEN
The structure of sialic acid on living cells can be modulated by metabolism of unnatural biosynthetic precursors. Here we investigate the conversion of a panel of azide-functionalized mannosamine and glucosamine derivatives into cell-surface sialosides. A key tool in this study is the Staudinger ligation, a highly selective reaction between modified triarylphosphines and azides that produces an amide-linked product. A preliminary study of the mechanism of this reaction, and refined conditions for its in vivo execution, are reported. The reaction provided a means to label the glycoconjugate-bound azidosugars with biochemical probes. Finally, we demonstrate that the cell-surface Staudinger ligation is compatible with hydrazone formation from metabolically introduced ketones. These two strategies provide a means to selectively modify cell-surface glycans with exogenous probes.