RESUMEN
Overexpression and aggregation of α-synuclein (ASyn) are linked to the onset and pathology of Parkinson's disease and related synucleinopathies. Elevated levels of the stress-induced chaperone Hsp70 protect against ASyn misfolding and ASyn-driven neurodegeneration in cell and animal models, yet there is minimal mechanistic understanding of this important protective pathway. It is generally assumed that Hsp70 binds to ASyn using its canonical and promiscuous substrate-binding cleft to limit aggregation. Here we report that this activity is due to a novel and unexpected mode of Hsp70 action, involving neither ATP nor the typical substrate-binding cleft. We use novel ASyn oligomerization assays to show that Hsp70 directly blocks ASyn oligomerization, an early event in ASyn misfolding. Using truncations, mutations, and inhibitors, we confirm that Hsp70 interacts with ASyn via an as yet unidentified, noncanonical interaction site in the C-terminal domain. Finally, we report a biological role for a similar mode of action in H4 neuroglioma cells. Together, these findings suggest that new chemical approaches will be required to target the Hsp70-ASyn interaction in synucleinopathies. Such approaches are likely to be more specific than targeting Hsp70's canonical action. Additionally, these results raise the question of whether other misfolded proteins might also engage Hsp70 via the same noncanonical mechanism.
Asunto(s)
Adenosina Trifosfato/metabolismo , Glioma/patología , Proteínas HSP70 de Choque Térmico/metabolismo , Agregación Patológica de Proteínas , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Glioma/genética , Glioma/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Humanos , Mutación , Células Tumorales CultivadasAsunto(s)
Dípteros , Intestinos , Zinc , Animales , Ingestión de Alimentos , Humanos , Intestinos/fisiologíaRESUMEN
Labeling proteins with high specificity and efficiency is a fundamental prerequisite for microscopic visualization of subcellular protein structures and interactions. Although the comparatively small size of epitope tags makes them less perturbative to fusion proteins, they require the use of large antibodies that often limit probe accessibility and effective resolution. Here we use the covalent SpyTag-SpyCatcher system as an epitope-like tag for fluorescent labeling of intracellular proteins in fixed cells for both conventional and super-resolution microscopy. We also applied this method to endogenous proteins by gene editing, demonstrating its high labeling efficiency and capability for isoform-specific labeling.
Asunto(s)
Adhesinas Bacterianas/química , Proteínas Portadoras/química , Fragmentos de Péptidos/química , Péptidos/química , Actinas/química , Adhesinas Bacterianas/metabolismo , Carbocianinas/química , Proteínas Portadoras/metabolismo , Cadenas Ligeras de Clatrina/química , Cadenas Ligeras de Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Colorantes Fluorescentes , Edición Génica , Células HeLa , Humanos , Queratinas/química , Microscopía Fluorescente , Fragmentos de Péptidos/metabolismo , Canales de Translocación SEC/química , Canales de Translocación SEC/metabolismo , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Protein Kinase A (PKA) is regulated spatially and temporally via scaffolding of its catalytic (Cα/ß) and regulatory (RI/RII) subunits by the A-kinase-anchoring proteins (AKAP). PKA engages in poorly understood interactions with autophagy, a key degradation pathway for neuronal cell homeostasis, partly via its AKAP11 scaffold. Mutations in AKAP11 drive schizophrenia and bipolar disorders (SZ-BP) through unknown mechanisms. Through proteomic-based analysis of immunopurified lysosomes, we identify the Cα-RIα-AKAP11 holocomplex as a prominent autophagy-associated protein kinase complex. AKAP11 scaffolds Cα-RIα to the autophagic machinery via its LC3-interacting region (LIR), enabling both PKA regulation by upstream signals, and its autophagy-dependent degradation. We identify Ser83 on the RIα linker-hinge region as an AKAP11-dependent phospho-residue that modulates RIα-Cα binding and cAMP-induced PKA activation. Decoupling AKAP11-PKA from autophagy alters Ser83 phosphorylation, supporting an autophagy-dependent checkpoint for PKA signaling. Ablating AKAP11 in induced pluripotent stem cell-derived neurons reveals dysregulation of multiple pathways for neuronal homeostasis. Thus, the autophagosome is a novel platform that modulate PKA signaling, providing a possible mechanistic link to SZ/BP pathophysiology.
RESUMEN
Lysosomes coordinate cellular metabolism and growth upon sensing of essential nutrients, including cholesterol. Through bioinformatic analysis of lysosomal proteomes, we identified lysosomal cholesterol signaling (LYCHOS, previously annotated as G protein-coupled receptor 155), a multidomain transmembrane protein that enables cholesterol-dependent activation of the master growth regulator, the protein kinase mechanistic target of rapamycin complex 1 (mTORC1). Cholesterol bound to the amino-terminal permease-like region of LYCHOS, and mutating this site impaired mTORC1 activation. At high cholesterol concentrations, LYCHOS bound to the GATOR1 complex, a guanosine triphosphatase (GTPase)-activating protein for the Rag GTPases, through a conserved cytoplasm-facing loop. By sequestering GATOR1, LYCHOS promotes cholesterol- and Rag-dependent recruitment of mTORC1 to lysosomes. Thus, LYCHOS functions in a lysosomal pathway for cholesterol sensing and couples cholesterol concentrations to mTORC1-dependent anabolic signaling.
Asunto(s)
Colesterol , Lisosomas , Diana Mecanicista del Complejo 1 de la Rapamicina , Receptores Acoplados a Proteínas G , Colesterol/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteoma/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
The centrosome serves as the main microtubule-organizing center in metazoan cells, yet despite its functional importance, little is known mechanistically about the structure and organizational principles that dictate protein organization in the centrosome. In particular, the protein-protein interactions that allow for the massive structural transition between the tightly organized interphase centrosome and the highly expanded matrix-like arrangement of the mitotic centrosome have been largely uncharacterized. Among the proteins that undergo a major transition is the Drosophila melanogaster protein centrosomin that contains a conserved carboxyl terminus motif, CM2. Recent crystal structures have shown this motif to be dimeric and capable of forming an intramolecular interaction with a central region of centrosomin. Here we use a combination of in-cell microscopy and in vitro oligomer assessment to show that dimerization is not necessary for CM2 recruitment to the centrosome and that CM2 alone undergoes significant cell cycle dependent rearrangement. We use NMR binding assays to confirm this intramolecular interaction and show that residues involved in solution are consistent with the published crystal structure and identify L1137 as critical for binding. Additionally, we show for the first time an in vitro interaction of CM2 with the Drosophila pericentrin-like-protein that exploits the same set of residues as the intramolecular interaction. Furthermore, NMR experiments reveal a calcium sensitive interaction between CM2 and calmodulin. Although unexpected because of sequence divergence, this suggests that centrosomin-mediated assemblies, like the mammalian pericentrin, may be calcium regulated. From these results, we suggest an expanded model where during interphase CM2 interacts with pericentrin-like-protein to form a layer of centrosomin around the centriole wall and that at the onset of mitosis this population acts as a nucleation site of intramolecular centrosomin interactions that support the expansion into the metaphase matrix.
Asunto(s)
Ciclo Celular/fisiología , Proteínas de Drosophila/metabolismo , Proteínas de Homeodominio/metabolismo , Animales , Sitios de Unión , Proteínas de Drosophila/fisiología , Drosophila melanogaster , Proteínas de Homeodominio/fisiología , Resonancia Magnética Nuclear Biomolecular , Reacción en Cadena de la Polimerasa , Unión Proteica , Técnicas del Sistema de Dos HíbridosRESUMEN
Messenger RNA (mRNA) plays a critical role in cellular growth and development. However, there have been limited methods available to visualize endogenous mRNA in living cells with ease. We have designed RNA-based fluorescence "turn-on" probes that target mRNA by fusing an unstable form of Spinach with target-complementary sequences. These probes have been demonstrated to be selective, stable, and capable of targeting various mRNAs for live E. coli imaging.
Asunto(s)
Aptámeros de Nucleótidos/química , Compuestos de Bencilo/química , Colorantes Fluorescentes/química , Imidazolinas/química , ARN Mensajero/análisis , Escherichia coli/química , Escherichia coli/genética , G-Cuádruplex , Hibridación de Ácido Nucleico , ARN Mensajero/química , Espectrometría de FluorescenciaRESUMEN
Photoactivatable fluorescent proteins (PA-FPs) are widely used in live single-molecule super-resolution imaging but emit substantially fewer photons than organic dyes do. Herein, we show that in heavy water (D2O) instead of H2O, common PA-FPs emit 26-54% more photons, effectively improving the localization precision in super-resolution imaging.
Asunto(s)
Óxido de Deuterio/química , Colorantes Fluorescentes/química , Proteínas Luminiscentes/química , Imagen Molecular/métodos , FotonesRESUMEN
Ruthenium diimine complexes have previously been used to facilitate light-activated electron transfer in the study of redox metalloproteins. Excitation at 488 nm leads to a photoexcited state, in which the complex can either accept or donate an electron, respectively, in the presence of a soluble sacrificial reductant or oxidant. Here, we describe a novel application of these complexes in mediating light-induced changes in cellular electrical activity. We demonstrate that RubpyC17 ([Ru(bpy)(2)(bpy-C17)](2+), where bpy is 2,2'-bipyridine and bpy-C17 is 2,2'-4-heptadecyl-4'-methyl-bipyridine), readily incorporates into the plasma membrane of cells, as evidenced by membrane-confined luminescence. Excitable cells incubated in RubpyC17 and then illuminated at 488 nm in the presence of the reductant ascorbate undergo membrane depolarization leading to firing of action potentials. In contrast, the same experiment performed with the oxidant ferricyanide, instead of ascorbate, leads to hyperpolarization. These experiments suggest that illumination of membrane-associated RubpyC17 in the presence of ascorbate alters the cell membrane potential by increasing the negative charge on the outer face of the cell membrane capacitor, effectively depolarizing the cell membrane. We rule out two alternative explanations for light-induced membrane potential changes, using patch clamp experiments: (1) light-induced direct interaction of RubpyC17 with ion channels and (2) light-induced membrane perforation. We show that incorporation of RubpyC17 into the plasma membrane of neuroendocrine cells enables light-induced secretion as monitored by amperometry. While the present work is focused on ruthenium diimine complexes, the findings point more generally to broader application of other transition metal complexes to mediate light-induced biological changes.