RESUMEN
All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, nonperturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.
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Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide and glycan motif and achieve cell-type selectivity via antigen-driven cell-surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in mouse models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific protein glycoforms on target cells.
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Mucinas , Neoplasias , Animales , Ratones , Mucinas/metabolismo , Péptido Hidrolasas/metabolismo , ProteolisisRESUMEN
Glycans are one of the fundamental biopolymers encountered in living systems. Compared to polynucleotide and polypeptide biosynthesis, polysaccharide biosynthesis is a uniquely combinatorial process to which interdependent enzymes with seemingly broad specificities contribute. The resulting intracellular cell surface, and secreted glycans play key roles in health and disease, from embryogenesis to cancer progression. The study and modulation of glycans in cell and organismal biology is aided by small molecule inhibitors of the enzymes involved in glycan biosynthesis. In this review, we survey the arsenal of currently available inhibitors, focusing on agents which have been independently validated in diverse systems. We highlight the utility of these inhibitors and drawbacks to their use, emphasizing the need for innovation for basic research as well as for therapeutic applications.
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Mucins are functionally implicated in a range of human pathologies, including cystic fibrosis, influenza, bacterial endocarditis, gut dysbiosis, and cancer. These observations have motivated the study of mucin biosynthesis as well as the development of strategies for inhibition of mucin glycosylation. Mammalian pathways for mucin catabolism, however, have remained underexplored. The canonical view, derived from analysis of N-glycoproteins in human lysosomal storage disorders, is that glycan degradation and proteolysis occur sequentially. Here, we challenge this view by providing genetic and biochemical evidence supporting mammalian proteolysis of heavily O-glycosylated mucin domains without prior deglycosylation. Using activity screening coupled with mass spectrometry, we ascribed mucin-degrading activity in murine liver to the lysosomal protease cathepsin D. Glycoproteomics of substrates digested with purified human liver lysosomal cathepsin D provided direct evidence for proteolysis within densely O-glycosylated domains. Finally, knockout of cathepsin D in a murine model of the human lysosomal storage disorder neuronal ceroid lipofuscinosis 10 resulted in accumulation of mucins in liver-resident macrophages. Our findings imply that mucin-degrading activity is a component of endogenous pathways for glycoprotein catabolism in mammalian tissues.
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Catepsina D , Lisosomas , Mucinas , Animales , Catepsina D/genética , Catepsina D/metabolismo , Glicoproteínas/metabolismo , Humanos , Lisosomas/enzimología , Mamíferos/metabolismo , Ratones , Mucinas/metabolismo , Polisacáridos/metabolismoRESUMEN
Mucin domains are densely O-glycosylated modular protein domains found in various extracellular and transmembrane proteins. Mucin-domain glycoproteins play important roles in many human diseases, such as cancer and cystic fibrosis, but the scope of the mucinome remains poorly defined. Recently, we characterized a bacterial O-glycoprotease, StcE, and demonstrated that an inactive point mutant retains binding selectivity for mucin-domain glycoproteins. In this work, we leverage inactive StcE to selectively enrich and identify mucin-domain glycoproteins from complex samples like cell lysate and crude ovarian cancer patient ascites fluid. Our enrichment strategy is further aided by an algorithm to assign confidence to mucin-domain glycoprotein identifications. This mucinomics platform facilitates detection of hundreds of glycopeptides from mucin domains and highly overlapping populations of mucin-domain glycoproteins from ovarian cancer patients. Ultimately, we demonstrate our mucinomics approach can reveal key molecular signatures of cancer from in vitro and ex vivo sources.
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Mucinas , Neoplasias Ováricas , Femenino , Glicopéptidos/química , Glicoproteínas/metabolismo , Glicosilación , Humanos , Mucinas/metabolismo , Neoplasias Ováricas/química , Neoplasias Ováricas/genéticaRESUMEN
The glycocalyx is a shell of heavily glycosylated proteins and lipids distributed on the cell surface of nearly all cell types. Recently, it has been found that bulky transmembrane glycoproteins such as MUC1 can modulate membrane shape by inducing membrane protrusions. In this work, we examine the reciprocal relationship of how membrane shape affects MUC1's spatial distribution on the cell membrane and its biological significance. By employing nanopatterned surfaces and membrane-sculpting proteins to manipulate membrane curvature, we show that MUC1 avoids positively-curved membranes (membrane invaginations) and accumulates on negatively-curved membranes (membrane protrusions). MUC1's curvature sensitivity is dependent on the length and the extent of glycosylation of its ectodomain, with large and highly glycosylated forms preferentially staying out of positive curvature. Interestingly, MUC1's avoidance of positive membrane curvature enables it to escape from endocytosis and being removed from the cell membrane. These findings also suggest that the truncation of MUC1's ectodomain, often observed in breast and ovarian cancers, may enhance its endocytosis and potentiate its intracellular accumulation and signaling.
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Endocitosis , Glicoproteínas , Membrana Celular , Fibras de la Dieta , Glicosilación , Proteínas de la MembranaRESUMEN
[This corrects the article DOI: 10.1016/j.mbplus.2022.100108.].
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Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammals use RNA as a third scaffold for glycosylation. Using a battery of chemical and biochemical approaches, we found that conserved small noncoding RNAs bear sialylated glycans. These "glycoRNAs" were present in multiple cell types and mammalian species, in cultured cells, and in vivo. GlycoRNA assembly depends on canonical N-glycan biosynthetic machinery and results in structures enriched in sialic acid and fucose. Analysis of living cells revealed that the majority of glycoRNAs were present on the cell surface and can interact with anti-dsRNA antibodies and members of the Siglec receptor family. Collectively, these findings suggest the existence of a direct interface between RNA biology and glycobiology, and an expanded role for RNA in extracellular biology.
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Membrana Celular/metabolismo , Polisacáridos/metabolismo , ARN/metabolismo , Animales , Anticuerpos/metabolismo , Secuencia de Bases , Vías Biosintéticas , Línea Celular , Supervivencia Celular , Humanos , Espectrometría de Masas , Ácido N-Acetilneuramínico/metabolismo , Poliadenilación , Polisacáridos/química , ARN/química , ARN/genética , ARN no Traducido/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Coloración y EtiquetadoRESUMEN
Glyco-immune checkpoint receptors, molecules that inhibit immune cell activity following binding to glycosylated cell-surface antigens, are emerging as attractive targets for cancer immunotherapy. Defining biologically relevant ligands that bind and activate such receptors, however, has historically been a significant challenge. Here, we present a CRISPRi genomic screening strategy that allowed unbiased identification of the key genes required for cell-surface presentation of glycan ligands on leukemia cells that bind the glyco-immune checkpoint receptors Siglec-7 and Siglec-9. This approach revealed a selective interaction between Siglec-7 and the mucin-type glycoprotein CD43. Further work identified a specific N-terminal glycopeptide region of CD43 containing clusters of disialylated O-glycan tetrasaccharides that form specific Siglec-7 binding motifs. Knockout or blockade of CD43 in leukemia cells relieves Siglec-7-mediated inhibition of immune killing activity. This work identifies a potential target for immune checkpoint blockade therapy and represents a generalizable approach to dissection of glycan-receptor interactions in living cells.
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Antígenos de Diferenciación Mielomonocítica/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Genoma Humano , Lectinas/metabolismo , Polisacáridos/metabolismo , Secuencias de Aminoácidos , Antígenos de Diferenciación Mielomonocítica/química , Línea Celular Tumoral , Membrana Celular/metabolismo , Glicopéptidos/metabolismo , Humanos , Sinapsis Inmunológicas/metabolismo , Células Asesinas Naturales/metabolismo , Lectinas/química , Leucosialina/química , Leucosialina/metabolismo , Ligandos , Unión ProteicaRESUMEN
Macrophages continuously survey their environment in search of pathogens or apoptotic corpses or debris. Targets intended for clearance expose ligands that initiate their phagocytosis ("eat me" signals), while others avoid phagocytosis by displaying inhibitory ligands ("don't eat me" signals). We report that such ligands can be obscured by the glycosaminoglycans and glycoproteins that coat pathogenic as well as malignant phagocytic targets. In addition, a reciprocal barrier of self-synthesized or acquired glycocalyx components on the macrophage surface shrouds phagocytic receptors, curtailing their ability to engage particles. The coating layers of macrophages and their targets hinder phagocytosis by both steric and electrostatic means. Their removal by enzymatic means is shown to markedly enhance phagocytic efficiency. In particular, we show that the removal of mucins, which are overexpressed in cancer cells, facilitates their clearance. These results shed light on the physical barriers that modulate phagocytosis, which have been heretofore underappreciated. VIDEO ABSTRACT.
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Candidiasis Invasiva/inmunología , Glicocálix/inmunología , Neoplasias/inmunología , Fagocitosis/inmunología , Adulto , Animales , Productos Biológicos/farmacología , Productos Biológicos/uso terapéutico , Antígeno CD47/antagonistas & inhibidores , Antígeno CD47/inmunología , Antígeno CD47/metabolismo , Candida albicans/inmunología , Candida albicans/metabolismo , Candidiasis Invasiva/microbiología , Modelos Animales de Enfermedad , Femenino , Glicocálix/metabolismo , Glicosaminoglicanos/metabolismo , Voluntarios Sanos , Humanos , Ácido Hialurónico/metabolismo , Inmunoglobulina G/farmacología , Inmunoglobulina G/uso terapéutico , Células MCF-7 , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Mucinas/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Peritoneo/inmunología , Peritoneo/microbiología , Fagocitos/efectos de los fármacos , Fagocitos/inmunología , Fagocitos/metabolismo , Fagocitosis/efectos de los fármacos , Cultivo Primario de Células , Células RAW 264.7 , Líquido Sinovial/citología , Líquido Sinovial/inmunología , Líquido Sinovial/metabolismo , Adulto JovenRESUMEN
Densely O-glycosylated mucin domains are found in a broad range of cell surface and secreted proteins, where they play key physiological roles. In addition, alterations in mucin expression and glycosylation are common in a variety of human diseases, such as cancer, cystic fibrosis, and inflammatory bowel diseases. These correlations have been challenging to uncover and establish because tools that specifically probe mucin domains are lacking. Here, we present a panel of bacterial proteases that cleave mucin domains via distinct peptide- and glycan-based motifs, generating a diverse enzymatic toolkit for mucin-selective proteolysis. By mutating catalytic residues of two such enzymes, we engineered mucin-selective binding agents with retained glycoform preferences. StcEE447D is a pan-mucin stain derived from enterohemorrhagic Escherichia coli that is tolerant to a wide range of glycoforms. BT4244E575A derived from Bacteroides thetaiotaomicron is selective for truncated, asialylated core 1 structures commonly associated with malignant and premalignant tissues. We demonstrated that these catalytically inactive point mutants enable robust detection and visualization of mucin-domain glycoproteins by flow cytometry, Western blot, and immunohistochemistry. Application of our enzymatic toolkit to ascites fluid and tissue slices from patients with ovarian cancer facilitated characterization of patients based on differences in mucin cleavage and expression patterns.
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Mucinas/análisis , Polisacárido Liasas/metabolismo , Adenocarcinoma/química , Secuencias de Aminoácidos , Western Blotting , Femenino , Citometría de Flujo , Humanos , Neoplasias Ováricas/química , Mutación Puntual , Polisacárido Liasas/química , Polisacárido Liasas/genéticaRESUMEN
The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein-for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins-the products of 40% of all protein-encoding genes7-are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified-but essential-component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.
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Espacio Extracelular/metabolismo , Lisosomas/metabolismo , Proteínas de la Membrana/metabolismo , Proteolisis , Proteínas Recombinantes de Fusión/metabolismo , Animales , Anticuerpos/química , Anticuerpos/metabolismo , Antígenos CD/metabolismo , Apolipoproteína E4/metabolismo , Antígeno B7-H1/metabolismo , Sistemas CRISPR-Cas , Línea Celular , Receptores ErbB/metabolismo , Femenino , Glicopéptidos/síntesis química , Glicopéptidos/metabolismo , Humanos , Ligandos , Proteínas de la Membrana/química , Ratones , Dominios Proteicos , Transporte de Proteínas , Receptor IGF Tipo 2/metabolismo , Receptores de Transferrina/metabolismo , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/química , Solubilidad , Especificidad por SustratoRESUMEN
Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.
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Glicosiltransferasas/metabolismo , Polisacáridos/metabolismo , Ingeniería de Proteínas/métodos , Vías Biosintéticas , Membrana Celular/metabolismo , Glicosilación , Glicosiltransferasas/química , Glicosiltransferasas/fisiología , Células HEK293 , Células Hep G2 , Humanos , Células K562 , N-Acetilgalactosaminiltransferasas/química , N-Acetilgalactosaminiltransferasas/metabolismo , N-Acetilgalactosaminiltransferasas/fisiología , Polisacáridos/química , Proteínas/metabolismo , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx. Mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface. A polymer brush model of the glycocalyx successfully predicts the effects of polymer size and cell-surface density on membrane morphologies. Specific glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled membrane structures and drive secretion of extracellular vesicles. Together, our results suggest a fundamental role for the glycocalyx in regulating curved membrane features that serve in communication between cells and with the extracellular matrix.
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Forma de la Célula , Matriz Extracelular/metabolismo , Glicocálix/metabolismo , Glicoproteínas de Membrana/metabolismo , Mucinas/metabolismo , Animales , Línea Celular , Matriz Extracelular/genética , Glicocálix/genética , Caballos , Humanos , Glicoproteínas de Membrana/genética , Mucinas/genéticaRESUMEN
The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Despite its relevance in both health and disease, studies of the glycocalyx remain hampered by a paucity of methods to spatially classify its components. We combine metabolic labeling, bioorthogonal chemistry, and super-resolution localization microscopy to image two constituents of cell-surface glycans, N-acetylgalactosamine (GalNAc) and sialic acid, with 10-20 nm precision in 2D and 3D. This approach enables two measurements: glycocalyx height and the distribution of individual sugars distal from the membrane. These measurements show that the glycocalyx exhibits nanoscale organization on both cell lines and primary human tumor cells. Additionally, we observe enhanced glycocalyx height in response to epithelial-to-mesenchymal transition and to oncogenic KRAS activation. In the latter case, we trace increased height to an effector gene, GALNT7. These data highlight the power of advanced imaging methods to provide molecular and functional insights into glycocalyx biology.
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Carcinoma Ductal Pancreático/patología , Glicocálix/metabolismo , Microscopía/métodos , N-Acetilgalactosaminiltransferasas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Pancreáticas/patología , Polisacáridos/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Transición Epitelial-Mesenquimal , Femenino , Glicosilación , Humanos , Neoplasias Ováricas/metabolismo , Neoplasias Pancreáticas/metabolismo , Pronóstico , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
Mucin domains are densely O-glycosylated modular protein domains that are found in a wide variety of cell surface and secreted proteins. Mucin-domain glycoproteins are known to be key players in a host of human diseases, especially cancer, wherein mucin expression and glycosylation patterns are altered. Mucin biology has been difficult to study at the molecular level, in part, because methods to manipulate and structurally characterize mucin domains are lacking. Here, we demonstrate that secreted protease of C1 esterase inhibitor (StcE), a bacterial protease from Escherichia coli, cleaves mucin domains by recognizing a discrete peptide- and glycan-based motif. We exploited StcE's unique properties to improve sequence coverage, glycosite mapping, and glycoform analysis of recombinant human mucins by mass spectrometry. We also found that StcE digests cancer-associated mucins from cultured cells and from ascites fluid derived from patients with ovarian cancer. Finally, using StcE, we discovered that sialic acid-binding Ig-type lectin-7 (Siglec-7), a glycoimmune checkpoint receptor, selectively binds sialomucins as biological ligands, whereas the related receptor Siglec-9 does not. Mucin-selective proteolysis, as exemplified by StcE, is therefore a powerful tool for the study of mucin domain structure and function.
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Antígenos CD/química , Antígenos de Diferenciación Mielomonocítica/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Lectinas/química , Metaloendopeptidasas/química , Mucinas/química , Proteínas de Neoplasias/química , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/química , Secuencias de Aminoácidos , Humanos , Espectrometría de Masas , Especificidad por SustratoRESUMEN
Metastasis depends upon cancer cell growth and survival within the metastatic niche. Tumors which remodel their glycocalyces, by overexpressing bulky glycoproteins like mucins, exhibit a higher predisposition to metastasize, but the role of mucins in oncogenesis remains poorly understood. Here we report that a bulky glycocalyx promotes the expansion of disseminated tumor cells in vivo by fostering integrin adhesion assembly to permit G1 cell cycle progression. We engineered tumor cells to display glycocalyces of various thicknesses by coating them with synthetic mucin-mimetic glycopolymers. Cells adorned with longer glycopolymers showed increased metastatic potential, enhanced cell cycle progression, and greater levels of integrin-FAK mechanosignaling and Akt signaling in a syngeneic mouse model of metastasis. These effects were mirrored by expression of the ectodomain of cancer-associated mucin MUC1. These findings functionally link mucinous proteins with tumor aggression, and offer a new view of the cancer glycocalyx as a major driver of disease progression.
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Carcinogénesis , Ciclo Celular , Proliferación Celular , Glicocálix/metabolismo , Neoplasias Mamarias Animales/secundario , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Glicocálix/genética , Humanos , Ratones , Mucina-1/genética , Mucina-1/metabolismoRESUMEN
Although purification of biotinylated molecules is highly efficient, identifying specific sites of biotinylation remains challenging. We show that anti-biotin antibodies enable unprecedented enrichment of biotinylated peptides from complex peptide mixtures. Live-cell proximity labeling using APEX peroxidase followed by anti-biotin enrichment and mass spectrometry yielded over 1,600 biotinylation sites on hundreds of proteins, an increase of more than 30-fold in the number of biotinylation sites identified compared to streptavidin-based enrichment of proteins.
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Anticuerpos/metabolismo , Biotina/metabolismo , Péptidos/química , Proteínas/química , Biotecnología/métodos , Biotinilación , Cromatografía Liquida , Células HEK293 , Humanos , Células Jurkat , Proteínas/aislamiento & purificación , Coloración y Etiquetado , Estreptavidina/metabolismo , Espectrometría de Masas en TándemRESUMEN
This protocol describes a method to obtain spatially resolved proteomic maps of specific compartments within living mammalian cells. An engineered peroxidase, APEX2, is genetically targeted to a cellular region of interest. Upon the addition of hydrogen peroxide for 1 min to cells preloaded with a biotin-phenol substrate, APEX2 generates biotin-phenoxyl radicals that covalently tag proximal endogenous proteins. Cells are then lysed, and biotinylated proteins are enriched with streptavidin beads and identified by mass spectrometry. We describe the generation of an appropriate APEX2 fusion construct, proteomic sample preparation, and mass spectrometric data acquisition and analysis. A two-state stable isotope labeling by amino acids in cell culture (SILAC) protocol is used for proteomic mapping of membrane-enclosed cellular compartments from which APEX2-generated biotin-phenoxyl radicals cannot escape. For mapping of open cellular regions, we instead use a 'ratiometric' three-state SILAC protocol for high spatial specificity. Isotopic labeling of proteins takes 5-7 cell doublings. Generation of the biotinylated proteomic sample takes 1 d, acquiring the mass spectrometric data takes 2-5 d and analysis of the data to obtain the final proteomic list takes 1 week.
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ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Proteoma/análisis , Proteómica/métodos , Biotina/metabolismo , Biotinilación , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Endonucleasas , Células HEK293 , Humanos , Peróxido de Hidrógeno/metabolismo , Marcaje Isotópico/métodos , Espectrometría de Masas , Enzimas Multifuncionales , Ingeniería de Proteínas , Proteoma/metabolismoRESUMEN
BACKGROUND: It is unclear whether long-term seizure outcomes in children are similar to those in adult epilepsy surgery patients. OBJECTIVE: To determine 5-year outcomes and antiepilepsy drug (AED) use in pediatric epilepsy surgery patients from a single institution. METHODS: The cohort consisted of children younger than 18 years of age whose 5-year outcome data would have been available by 2010. Comparisons were made between patients with and without 5-year data (n = 338), patients with 5-year data for seizure outcome (n = 257), and seizure-free patients on and off AEDs (n = 137). RESULTS: Five-year data were available from 76% of patients. More seizure-free patients with focal resections for hippocampal sclerosis and tumors lacked 5-year data compared with other cases. Of those with 5-year data, 53% were continuously seizure free, 18% had late seizure recurrence, 3% became seizure free after initial failure, and 25% were never seizure free. Patients were more likely to be continuously seizure free if their surgery was performed during the period 2001 to 2005 (68%) compared with surgery performed from 1996 to 2000 (61%), 1991 to 1995 (36%), and 1986 to 1990 (46%). More patients had 1 or fewer seizures per month in the late seizure recurrence (47%) compared with the not seizure-free group (20%). Four late deaths occurred in the not seizure-free group compared with 1 in the seizure-free group. Of patients who were continuously seizure free, 55% were not taking AEDs, and more cortical dysplasia patients (74%) had stopped taking AEDs compared with hemimegalencephaly patients (18%). CONCLUSION: In children, 5-year outcomes improved over 20 years of clinical experience. Our results are similar to those of adult epilepsy surgery patients despite mostly extratemporal and hemispheric operations for diverse developmental etiologies.