RESUMEN
The neurofilament (NF) cytoskeleton is critical for neuronal morphology and function. In particular, the neurofilament-light (NF-L) subunit is required for NF assembly in vivo and is mutated in subtypes of Charcot-Marie-Tooth (CMT) disease. NFs are highly dynamic, and the regulation of NF assembly state is incompletely understood. Here, we demonstrate that human NF-L is modified in a nutrient-sensitive manner by O-linked-ß-N-acetylglucosamine (O-GlcNAc), a ubiquitous form of intracellular glycosylation. We identify five NF-L O-GlcNAc sites and show that they regulate NF assembly state. NF-L engages in O-GlcNAc-mediated protein-protein interactions with itself and with the NF component α-internexin, implying that O-GlcNAc may be a general regulator of NF architecture. We further show that NF-L O-GlcNAcylation is required for normal organelle trafficking in primary neurons. Finally, several CMT-causative NF-L mutants exhibit perturbed O-GlcNAc levels and resist the effects of O-GlcNAcylation on NF assembly state, suggesting a potential link between dysregulated O-GlcNAcylation and pathological NF aggregation. Our results demonstrate that site-specific glycosylation regulates NF-L assembly and function, and aberrant NF O-GlcNAcylation may contribute to CMT and other neurodegenerative disorders.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Humanos , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/patología , Filamentos Intermedios , Mutación , Glicosilación , Acetilglucosamina , Procesamiento Proteico-PostraduccionalRESUMEN
The neurofilament (NF) cytoskeleton is critical for neuronal morphology and function. In particular, the neurofilament-light (NF-L) subunit is required for NF assembly in vivo and is mutated in subtypes of Charcot-Marie-Tooth (CMT) disease. NFs are highly dynamic, and the regulation of NF assembly state is incompletely understood. Here, we demonstrate that human NF-L is modified in a nutrient-sensitive manner by O-linked-ß-N-acetylglucosamine (O-GlcNAc), a ubiquitous form of intracellular glycosylation. We identify five NF-L O-GlcNAc sites and show that they regulate NF assembly state. Interestingly, NF-L engages in O-GlcNAc-mediated protein-protein interactions with itself and with the NF component α-internexin, implying that O-GlcNAc is a general regulator of NF architecture. We further show that NF-L O-GlcNAcylation is required for normal organelle trafficking in primary neurons, underlining its functional significance. Finally, several CMT-causative NF-L mutants exhibit perturbed O-GlcNAc levels and resist the effects of O-GlcNAcylation on NF assembly state, indicating a potential link between dysregulated O-GlcNAcylation and pathological NF aggregation. Our results demonstrate that site-specific glycosylation regulates NF-L assembly and function, and aberrant NF O-GlcNAcylation may contribute to CMT and other neurodegenerative disorders.
RESUMEN
O-linked ß-N-acetylglucosamine (O-GlcNAc) is a dynamic form of intracellular glycosylation common in animals, plants and other organisms. O-GlcNAcylation is essential in mammalian cells and is dysregulated in myriad human diseases, such as cancer, neurodegeneration and metabolic syndrome. Despite this pathophysiological significance, key aspects of O-GlcNAc signaling remain incompletely understood, including its impact on fundamental cell biological processes. Here, we investigate the role of O-GlcNAcylation in the coat protein II complex (COPII), a system universally conserved in eukaryotes that mediates anterograde vesicle trafficking from the endoplasmic reticulum. We identify new O-GlcNAcylation sites on Sec24C, Sec24D and Sec31A, core components of the COPII system, and provide evidence for potential nutrient-sensitive pathway regulation through site-specific glycosylation. Our work suggests a new connection between metabolism and trafficking through the conduit of COPII protein O-GlcNAcylation.
Asunto(s)
Acetilglucosamina , Retículo Endoplásmico , Acetilglucosamina/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Glicosilación , Mamíferos/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Nutrientes , Procesamiento Proteico-Postraduccional , Transducción de SeñalRESUMEN
Gigaxonin (also known as KLHL16) is an E3 ligase adaptor protein that promotes the ubiquitination and degradation of intermediate filament (IF) proteins. Mutations in human gigaxonin cause the fatal neurodegenerative disease giant axonal neuropathy (GAN), in which IF proteins accumulate and aggregate in axons throughout the nervous system, impairing neuronal function and viability. Despite this pathophysiological significance, the upstream regulation and downstream effects of normal and aberrant gigaxonin function remain incompletely understood. Here, we report that gigaxonin is modified by
Asunto(s)
Acetilglucosamina/metabolismo , Proteínas del Citoesqueleto/metabolismo , Neuropatía Axonal Gigante/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Antígenos de Neoplasias/metabolismo , Sitios de Unión , Línea Celular , Proteínas del Citoesqueleto/genética , Epigénesis Genética , Terapia Genética , Neuropatía Axonal Gigante/etiología , Neuropatía Axonal Gigante/genética , Neuropatía Axonal Gigante/terapia , Glicosilación , Histona Acetiltransferasas/metabolismo , Humanos , Hialuronoglucosaminidasa/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Modelos Biológicos , Estado Nutricional , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteostasis , Serina/metabolismo , Treonina/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Proteins smaller than about 50 kDa are currently too small to be imaged at high resolution by cryo-electron microscopy (cryo-EM), leaving most protein molecules in the cell beyond the reach of this powerful structural technique. Here we use a designed protein scaffold to bind and symmetrically display 12 copies of a small 26 kDa protein, green fluorescent protein (GFP). We show that the bound cargo protein is held rigidly enough to visualize it at a resolution of 3.8 Å by cryo-EM, where specific structural features of the protein are visible. The designed scaffold is modular and can be modified through modest changes in its amino acid sequence to bind and display diverse proteins for imaging, thus providing a general method to break through the lower size limitation in cryo-EM.