The AD tau core spontaneously self-assembles and recruits full-length tau to filaments.
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ABSTRACT:
SUBMITTER: Carlomagno Y
PROVIDER: S-EPMC8094113 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Tau is a neuronal protein that stabilizes the microtubule (MT) network, but it also forms filaments associated with Alzheimer's disease. Understanding Tau-MT and Tau-Tau interactions would help to establish Tau function in health and disease. For many years, literature reports on Tau-MT binding behavior and affinity have remained surprisingly contradictory (e.g., 10-fold variation in Tau-MT affinity). Tau-Tau interactions have also been investigated, but whether MTs might affect Tau filament formation is unknown. We have addressed these issues through binding assays and microscopy. We assessed Tau-MT interactions via cosedimentation and found that the measured affinity of Tau varies greatly, depending on the experimental design and the protein concentrations used. To investigate this dependence, we used fluorescence microscopy to examine Tau-MT binding. Strikingly, we found that Taxol-stabilized MTs promote Tau filament formation without characterized Tau-filament inducers. We propose that these novel Tau filaments account for the incongruence in Tau-MT affinity measurements. Moreover, electron microscopy reveals that these filaments appear similar to the heparin-induced Alzheimer's model. These observations suggest that the MT-induced Tau filaments provide a new model for Alzheimer's studies and that MTs might play a role in the formation of Alzheimer's-associated neurofibrillary tangles.
| S-EPMC3521687 | biostudies-literature
Project description: Not available
| EMPIAR-11750 | biostudies-other
Project description:The tau protein is a highly soluble and natively unfolded protein. Under pathological conditions, tau undergoes multiple post-translational modifications (PTMs) and conformational changes to form insoluble filaments, which are the proteinaceous signatures of tauopathies. To dissect the crosstalk among tau PTMs during the aggregation process, we phosphorylated and ubiquitylated recombinant tau in vitro using GSK3β and CHIP, respectively. The resulting phospho-ub-tau contained conventional polyubiquitin chains with lysine 48 linkages, sufficient for proteasomal degradation, whereas unphosphorylated ub-tau species retained only one-three ubiquitin moieties. Mass-spectrometric analysis of in vitro reconstituted phospho-ub-tau revealed seven additional ubiquitylation sites, some of which are known to stabilize tau protofilament stacking in the human brain with tauopathy. When the ubiquitylation reaction was prolonged, phospho-ub-tau transformed into insoluble hyperubiquitylated tau species featuring fibrillar morphology and in vitro seeding activity. We developed a small-molecule inhibitor of CHIP through biophysical screening; this effectively suppressed tau ubiquitylation in vitro and delayed its aggregation in cultured cells including primary cultured neurons. Our biochemical findings point to a "multiple-hit model," where sequential events of tau phosphorylation and hyperubiquitylation function as a key driver of the fibrillization process, thus indicating that targeting tau ubiquitylation may be an effective strategy to alleviate the course of tauopathies.
| S-EPMC8179656 | biostudies-literature
Project description:Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell. Emerging evidence indicates that nonglycolytic metabolic enzymes regulating diverse pathways can assemble into polymers. We now show tetramer- and substrate-dependent filament assembly by phosphofructokinase-1 (PFK1), which is considered the "gatekeeper" of glycolysis because it catalyzes the step committing glucose to breakdown. Recombinant liver PFK1 (PFKL) isoform, but not platelet PFK1 (PFKP) or muscle PFK1 (PFKM) isoforms, assembles into filaments. Negative-stain electron micrographs reveal that filaments are apolar and made of stacked tetramers oriented with exposed catalytic sites positioned along the edge of the polymer. Electron micrographs and biochemical data with a PFKL/PFKP chimera indicate that the PFKL regulatory domain mediates filament assembly. Quantified live-cell imaging shows dynamic properties of localized PFKL puncta that are enriched at the plasma membrane. These findings reveal a new behavior of a key glycolytic enzyme with insights on spatial organization and isoform-specific glucose metabolism in cells.
| S-EPMC5551713 | biostudies-literature
Project description:Abundant tau inclusions are a defining hallmark of several human neurodegenerative diseases, including Alzheimer's disease. Protein fragmentation is a widely observed event in neurodegenerative proteinopathies. The relevance of tau fragmentation for the neurodegenerative process in tauopathies has yet remained unclear. Here we found that co-expression of truncated and full-length human tau in mice provoked the formation of soluble high-molecular-weight tau, the failure of axonal transport, clumping of mitochondria, disruption of the Golgi apparatus and missorting of synaptic proteins. This was associated with extensive nerve cell dysfunction and severe paralysis by the age of 3 weeks. When the expression of truncated tau was halted, most mice recovered behaviorally and functionally. In contrast, co-expression of full-length tau isoforms did not result in paralysis. Truncated tau thus induces extensive but reversible neurotoxicity in the presence of full-length tau through the formation of nonfilamentous high-molecular-weight tau aggregates, in the absence of tau filaments. Targeting tau fragmentation may provide a novel approach for the treatment of human tauopathies.
| S-EPMC5116481 | biostudies-literature
Project description:526-residue FUS functions to self-assemble into reversible droplets/hydrogels, which could be further solidified into pathological fibrils. FUS is intrinsically prone to aggregation, composed of N-terminal low-sequence complexity (LC); RNA-recognition motif (RRM) and C-terminal LC domains. Intriguingly, previous in vivo studies revealed that its RRM is required for manifesting FUS cytotoxicity but the underlying mechanism remains unknown. Here, we characterized solution conformations of FUS and its five differentially dissected fragments, followed by detailed investigations on thermal unfolding, NMR dynamics and self-assembly of RRM. The results decipher: (1) the N- and C-terminal LC domains are intrinsically disordered, while RRM is folded. Intriguingly, well-dispersed HSQC peaks of RRM disappear in the full-length FUS, reminiscent of the previous observation on TDP-43. (2) FUS RRM is characteristic of irreversible unfolding. "Model-free" analysis of NMR relaxation data decodes that RRM has high ps-ns conformational dynamics even over some residues within secondary structure regions. (3) RRM spontaneously self-assembles into amyloid fibrils. Therefore, in addition to the well-established prion-like region, FUS RRM is also prone to self-assembly to form amyloid fibrils. Taken together, FUS RRM appears to play a crucial role in exaggerating the physiological/reversible self-assembly into pathological/irreversible fibrillization, thus contributing to manifestation of FUS cytotoxicity.
| S-EPMC5430844 | biostudies-literature
Project description:The capsid shell of infectious hepatitis B virus (HBV) is composed of 240 copies of a single protein called HBV core antigen (HBc). An atomic model of a core assembled from truncated HBc was determined previously by X-ray crystallography. In an attempt to obtain atomic structural information of HBV core in a near native, non-crystalline environment, we reconstructed a 3.5Å-resolution structure of a recombinant core assembled from full-length HBc by cryo electron microscopy (cryoEM) and derived an atomic model. The structure shows that the 240 molecules of full-length HBc form a core with two layers. The outer layer, composed of the N-terminal assembly domain, is similar to the crystal structure of the truncated HBc, but has three differences. First, unlike the crystal structure, our cryoEM structure shows no disulfide bond between the Cys61 residues of the two subunits within the dimer building block, indicating such bond is not required for core formation. Second, our cryoEM structure reveals up to four more residues in the linker region (amino acids 140-149). Third, the loops in the cryoEM structures containing this linker region in subunits B and C are oriented differently (~30° and ~90°) from their counterparts in the crystal structure. The inner layer, composed of the C-terminal arginine-rich domain (ARD) and the ARD-bound RNAs, is partially-ordered and connected with the outer layer through linkers positioned around the two-fold axes. Weak densities emanate from the rims of positively charged channels through the icosahedral three-fold and local three-fold axes. We attribute these densities to the exposed portions of some ARDs, thus explaining ARD's accessibility by proteases and antibodies. Our data supports a role of ARD in mediating communication between inside and outside of the core during HBV maturation and envelopment.
| S-EPMC3765168 | biostudies-literature
Project description:BACKGROUND:Alzheimer's disease is associated with the accumulation of intracellular Tau tangles within neurons and extracellular amyloid-? plaques in the brain parenchyma, which altogether results in synaptic loss and neurodegeneration. Extracellular concentrations of oligomers and aggregated proteins initiate microglial activation and convert their state of synaptic surveillance into a destructive inflammatory state. Although Tau oligomers have fleeting nature, they were shown to mediate neurotoxicity and microglial pro-inflammation. Due to the instability of oligomers, in vitro experiments become challenging, and hence, the stability of the full-length Tau oligomers is a major concern. METHODS:In this study, we have prepared and stabilized hTau40WT oligomers, which were purified by size-exclusion chromatography. The formation of the oligomers was confirmed by western blot, thioflavin-S, 8-anilinonaphthaalene-1-sulfonic acid fluorescence, and circular dichroism spectroscopy, which determine the intermolecular cross-? sheet structure and hydrophobicity. The efficiency of N9 microglial cells to phagocytose hTau40WT oligomer and subsequent microglial activation was studied by immunofluorescence microscopy with apotome. The one-way ANOVA was performed for the statistical analysis of fluorometric assay and microscopic analysis. RESULTS:Full-length Tau oligomers were detected in heterogeneous globular structures ranging from 5 to 50?nm as observed by high-resolution transmission electron microscopy, which was further characterized by oligomer-specific A11 antibody. Immunocytochemistry studies for oligomer treatment were evidenced with A11+ Iba1high microglia, suggesting that the phagocytosis of extracellular Tau oligomers leads to microglial activation. Also, the microglia were observed with remodeled filopodia-like actin structures upon the exposure of oligomers and aggregated Tau. CONCLUSION:The peri-membrane polymerization of actin filament and co-localization of Iba1 relate to the microglial movements for phagocytosis. Here, these findings suggest that microglia modified actin cytoskeleton for phagocytosis and rapid clearance of Tau oligomers in Alzheimer's disease condition.
| S-EPMC6950897 | biostudies-literature
Project description:The presence of non-conserved peripheral elements in all naturally occurring group I introns underline their importance in ensuring the natural intron function. Recently, we reported that some peripheral elements are conserved in group I introns of IE subgroup. Using self-splicing activity as a readout, our initial screening revealed that one such conserved peripheral elements, P2.1, is mainly required to fold the catalytically active structure of the Candida ribozyme, an IE intron. Unexpectedly, the essential function of P2.1 resides in a sequence-conserved short stem of P2.1 but not in a long-range interaction associated with the loop of P2.1 that stabilizes the ribozyme structure. The P2.1 stem is indispensable in folding the compact ribozyme core, most probably by forming a triple helical interaction with two core helices, P3 and P6. Surprisingly, although the ribozyme lacking the P2.1 stem renders a loosely folded core and the loss of self-splicing activity requires two consecutive transesterifications, the mutant ribozyme efficiently catalyzes the first transesterification reaction. These results suggest that the intron self-splicing demands much more ordered structure than does one independent transesterification, highlighting that the universally present peripheral elements achieve their functional importance by enabling the highly ordered structure through diverse tertiary interactions.
| S-EPMC1185575 | biostudies-literature
Project description:A variety of genetic and biochemical evidence suggests that amyloid ? (A?) oligomers promote downstream errors in Tau action, in turn inducing neuronal dysfunction and cell death in Alzheimer and related dementias. To better understand molecular mechanisms involved in A?-mediated neuronal cell death, we have treated primary rat hippocampal cultures with A? oligomers and examined the resulting cellular changes occurring before and during the induction of cell death with a focus on altered Tau biochemistry. The most rapid neuronal responses upon A? administration are activation of caspase 3/7 and calpain proteases. A? also appears to reduce Akt and Erk1/2 kinase activities while increasing GSK3? and Cdk5 activities. Shortly thereafter, substantial Tau degradation begins, generating relatively stable Tau fragments. Only a very small fraction of full-length Tau remains intact after 4 h of A? treatment. In conflict with expectations based on suggested increases of GSK3? and Cdk5 activities, A? does not cause any major increases in phosphorylation of full-length Tau as assayed by immunoblotting one-dimensional gels with 11 independent site- and phospho-specific anti-Tau antibodies as well as by immunoblotting two-dimensional gels probed with a pan-Tau antibody. There are, however, subtle and transient increases in Tau phosphorylation at 3-4 specific sites before its degradation. Taken together, these data are consistent with the notion that A?-mediated neuronal cell death involves the loss of full-length Tau and/or the generation of toxic fragments but does not involve or require hyperphosphorylation of full-length Tau.
| S-EPMC3121465 | biostudies-literature