RESUMEN
Batten disease, the most prevalent form of neurodegeneration in children, is caused by mutations in the CLN3 gene, which encodes a lysosomal transmembrane protein. CLN3 loss leads to significant accumulation of glycerophosphodiesters (GPDs), the end products of glycerophospholipid catabolism in the lysosome. Despite GPD storage being robustly observed upon CLN3 loss, the role of GPDs in neuropathology remains unclear. Here, we demonstrate that GPDs act as potent inhibitors of glycerophospholipid catabolism in the lysosome using human cell lines and mouse models. Mechanistically, GPDs bind and competitively inhibit the lysosomal phospholipases PLA2G15 and PLBD2, which we establish to possess phospholipase B activity. GPDs effectively inhibit the rate-limiting lysophospholipase activity of these phospholipases. Consistently, lysosomes of CLN3-deficient cells and tissues accumulate toxic lysophospholipids. Our work establishes that the storage material in Batten disease directly disrupts lysosomal lipid homeostasis, suggesting GPD clearance as a potential therapeutic approach to this fatal disease.
Asunto(s)
Glicoproteínas de Membrana , Lipofuscinosis Ceroideas Neuronales , Ratones , Animales , Niño , Humanos , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Lipofuscinosis Ceroideas Neuronales/genética , Lipofuscinosis Ceroideas Neuronales/metabolismo , Lipofuscinosis Ceroideas Neuronales/patología , Lisosomas/metabolismo , Fosfolipasas/metabolismo , Glicerofosfolípidos/metabolismo , Fosfolípidos/metabolismoRESUMEN
Proteasomes are essential molecular machines responsible for the degradation of proteins in eukaryotic cells. Altered proteasome activity has been linked to neurodegeneration, auto-immune disorders and cancer. Despite the relevance for human disease and drug development, no method currently exists to monitor proteasome composition and interactions in vivo in animal models. To fill this gap, we developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and generated a new mouse model enabling the quantification of proteasome interactions by mass spectrometry. We show that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on their activity. We demonstrate the utility of our method by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling enables the identification of both endogenous and small-molecule-induced proteasome substrates.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Ratones , Humanos , Espectrometría de Masas/métodos , Mapeo de Interacción de ProteínasRESUMEN
Web spiders use specialized glands to produce silk proteins, so-called spidroins, which assemble into extraordinarily tough silk fibers through tightly regulated phase and structural transitions. A crucial step in the polymerization of spidroins is the pH-triggered assembly of their N-terminal domains (NTDs) into tight dimers. Major ampullate spidroin NTDs contain an unusually high content of the amino acid methionine. We previously showed that the simultaneous mutation of the six hydrophobic core methionine residues to leucine in the NTD of the major ampullate spidroin 1 from Euprosthenops australis, a nursery web spider, yields a protein (L6-NTD) retaining a three-dimensional fold identical to the wildtype (WT) domain, yet with a significantly increased stability. Further, the dynamics of the L6-NTD are significantly reduced and the ability to dimerize is severely impaired compared to the WT domain. These properties lead to significant changes in the NMR spectra between WT and L6-NTD so that the previously available WT-NTD assignments cannot be transferred to the mutant protein. Here, we thus report the de novo NMR backbone and side chain assignments of the major ampullate spidroin 1 L6-NTD variant from E. australis as a prerequisite for obtaining further insights into protein structure and dynamics.
Asunto(s)
Fibroínas/química , Resonancia Magnética Nuclear Biomolecular , Multimerización de Proteína , Arañas/metabolismo , Animales , Dominios Proteicos , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
Web spiders connect silk proteins, so-called spidroins, into fibers of extraordinary toughness. The spidroin N-terminal domain (NTD) plays a pivotal role in this process: it polymerizes spidroins through a complex mechanism of dimerization. Here we analyze sequences of spidroin NTDs and find an unusually high content of the amino acid methionine. We simultaneously mutate all methionines present in the hydrophobic core of a spidroin NTD from a nursery web spider's dragline silk to leucine. The mutated NTD is strongly stabilized and folds at the theoretical speed limit. The structure of the mutant is preserved, yet its ability to dimerize is substantially impaired. We find that side chains of core methionines serve to mobilize the fold, which can thereby access various conformations and adapt the association interface for tight binding. Methionine in a hydrophobic core equips a protein with the capacity to dynamically change shape and thus to optimize its function.
Asunto(s)
Fibroínas/metabolismo , Metionina/metabolismo , Seda/metabolismo , Arañas/metabolismo , Algoritmos , Secuencia de Aminoácidos , Animales , Fibroínas/química , Fibroínas/genética , Interacciones Hidrofóbicas e Hidrofílicas , Metionina/genética , Mutación , Conformación Proteica , Pliegue de Proteína , Multimerización de Proteína , Homología de Secuencia de Aminoácido , Seda/química , Espectrometría de Fluorescencia , Arañas/genética , TermodinámicaRESUMEN
T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (VL) or variable heavy (VH) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies.
Asunto(s)
Anticuerpos/farmacología , Antineoplásicos Inmunológicos/farmacología , Complejo CD3/metabolismo , Inmunoterapia/métodos , Linfocitos T/inmunología , Animales , Anticuerpos/genética , Antineoplásicos Inmunológicos/inmunología , Sitios de Unión , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Efecto Espectador , Línea Celular Tumoral , Femenino , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Humanos , Activación de Linfocitos , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Medicina de Precisión/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Web spiders synthesize silk fibers of unique strength and extensibility through the controlled self-assembly of protein building blocks, so-called spidroins. The spidroin C-terminal domain is highly conserved and connects two polypeptide chains through formation of an all-helical, intertwined dimer. Here we use contact-induced fluorescence self-quenching and resonance energy transfer in combination with far-UV circular dichroism spectroscopy as three orthogonal structural probes to dissect the mechanism of folding and dimerization of a spidroin C-terminal domain from the major ampullate gland of the nursery web spider Euprosthenops australis. We show that helices forming the dimer core assemble very rapidly and fold on association. Subsequently, peripheral helices fold and dock slowly onto the preformed core. Lability of outer helices facilitates formation of a highly expanded, partially folded dimer. The high end-to-end distance of chain termini in the partially folded dimer suggests an extensibility module that contributes to elasticity of spider silk.
Asunto(s)
Fibroínas/metabolismo , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Arañas , Animales , Dicroismo Circular , Dimerización , Fibroínas/ultraestructura , Transferencia Resonante de Energía de FluorescenciaRESUMEN
Web spiders synthesize silk fibres, nature's toughest biomaterial, through the controlled assembly of fibroin proteins, so-called spidroins. The highly conserved spidroin N-terminal domain (NTD) is a pH-driven self-assembly device that connects spidroins to super-molecules in fibres. The degree to which forces of self-assembly is conserved across spider glands and species is currently unknown because quantitative measures are missing. Here, we report the comparative investigation of spidroin NTDs originating from the major ampullate glands of the spider species Euprosthenops australis, Nephila clavipes, Latrodectus hesperus, and Latrodectus geometricus. We characterized equilibrium thermodynamics and kinetics of folding and self-association using dynamic light scattering, stopped-flow fluorescence and circular dichroism spectroscopy in combination with thermal and chemical denaturation experiments. We found cooperative two-state folding on a sub-millisecond time scale through a late transition state of all four domains. Stability was compromised by repulsive electrostatic forces originating from clustering of point charges on the NTD surface required for function. pH-driven dimerization proceeded with characteristic fast kinetics yielding high affinities. Results showed that energetics and kinetics of NTD self-assembly are highly conserved across spider species despite the different silk mechanical properties and web geometries they produce.