Molecular Deformation Is a Key Factor in Screening Aggregation Inhibitor for Intrinsically Disordered Protein Tau
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ABSTRACT:
SUBMITTER: Chai K
PROVIDER: S-EPMC10979476 | biostudies-literature | 2024 Mar
REPOSITORIES: biostudies-literature
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Project description:Tau is an intrinsically disordered protein (IDP) implicated in Alzheimer's disease. Recently, tau proteins were discovered to be able to catalyze self-acetylation, which may promote its pathological aggregation. Understanding the paradox of tau's random-like conformations, aggregation propensity, and enzymatic activity are challenging questions. We characterized the atomic structures of two truncated tau constructs, K18 and K19, consisting of, respectively, only the four- and three-repeats of tau protein, providing structural insights into tau's paradox. Extensive 4.8 μs replica-exchange molecular dynamics simulations of the tau proteins achieved quantitative correlation with experimental Cα chemical shifts. Our results revealed (1) dynamically ordered conformations with close lysine-cysteine distances essential for tau self-acetylation and (2) high β-sheet content and large hydrophobic surface exposure for the two critical hexapeptides (275VQIINK280 and 306VQIVYK311), crucial for tau aggregation. Together, they illuminate tau's perplexing behavior of how its disordered state can accomplish both roles.
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Project description:Intrinsically disordered proteins (IDPs) are a unique class of proteins that have no stable native structure, a feature that allows them to adopt a wide variety of extended and compact conformations that facilitate a large number of vital physiological functions. One of the most well-known IDPs is the microtubule-associated tau protein, which regulates microtubule growth in the nervous system. However, dysfunctions in tau can lead to tau oligomerization, fibril formation, and neurodegenerative disease, including Alzheimer's disease. Using a combination of simulations and experiments, we explore the role of osmolytes in regulating the conformation and aggregation propensities of the R2/wt peptide, a fragment of tau containing the aggregating paired helical filament (PHF6*). We show that the osmolytes urea and trimethylamine N-oxide (TMAO) shift the population of IDP monomer structures, but that no new conformational ensembles emerge. Although urea halts aggregation, TMAO promotes the formation of compact oligomers (including helical oligomers) through a newly proposed mechanism of redistribution of water around the perimeter of the peptide. We put forth a "superposition of ensembles" hypothesis to rationalize the mechanism by which IDP structure and aggregation is regulated in the cell.
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Project description:S100 proteins are small dimeric calcium-binding proteins which control cell cycle, growth and differentiation via interactions with different target proteins. Intrinsic disorder is a hallmark among many signaling proteins and S100 proteins have been proposed to contain disorder-prone regions. Interestingly, some S100 proteins also form amyloids: S100A8/A9 forms fibrils in prostatic inclusions and S100A6 fibrillates in vitro and seeds SOD1 aggregation. Here we report a study designed to investigate whether ?-aggregation is a feature extensive to more members of S100 family. In silico analysis of seven human S100 proteins revealed a direct correlation between aggregation and intrinsic disorder propensity scores, suggesting a relationship between these two independent properties. Averaged position-specific analysis and structural mapping showed that disorder-prone segments are contiguous to aggregation-prone regions and that whereas disorder is prominent on the hinge and target protein-interaction regions, segments with high aggregation propensity are found in ordered regions within the dimer interface. Acidic conditions likely destabilize the seven S100 studied by decreasing the shielding of aggregation-prone regions afforded by the quaternary structure. In agreement with the in silico analysis, hydrophobic moieties become accessible as indicated by strong ANS fluorescence. ATR-FTIR spectra support a structural inter-conversion from ?-helices to intermolecular ?-sheets, and prompt ThT-binding takes place with no noticeable lag phase. Dot blot analysis using amyloid conformational antibodies denotes a high diversity of conformers; subsequent analysis by TEM shows fibrils as dominant species. Altogether, our data suggests that ?-aggregation and disorder-propensity are related properties in S100 proteins, and that the onset of aggregation is likely triggered by loss of protective tertiary and quaternary interactions.
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Project description:Protein structure is determined by the amino acid sequence and a variety of post-translational modifications, and provides the basis for physiological properties. Not all proteins in the proteome attain a stable conformation; roughly one third of human proteins are unstructured or contain intrinsically disordered regions exceeding 40% of their length. Proteins comprising or containing extensive unstructured regions are termed intrinsically disordered proteins (IDPs). IDPs are known to be overrepresented in protein aggregates of diverse neurodegenerative diseases. We evaluated the importance of disordered proteins in the nematode Caenorhabditis elegans, by RNAi-mediated knockdown of IDPs in disease-model strains that mimic aggregation associated with neurodegenerative pathologies. Not all disordered proteins are sequestered into aggregates, and most of the tested aggregate-protein IDPs contribute to important physiological functions such as stress resistance or reproduction. Despite decades of research, we still do not understand what properties of a disordered protein determine its entry into aggregates. We have employed machine-learning models to identify factors that predict whether a disordered protein is found in sarkosyl-insoluble aggregates isolated from neurodegenerative-disease brains (both AD and PD). Machine-learning predictions, coupled with principal component analysis (PCA), enabled us to identify the physiochemical properties that determine whether a disordered protein will be enriched in neuropathic aggregates.
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Project description:Tau is an intrinsically disordered protein that has the ability to self-assemble to form paired helical and straight filaments in Alzheimer's disease, as well as the ability to form additional distinct tau filaments in other tauopathies. In the presence of microtubules, tau forms an elongated form associated with tubulin dimers via a series of imperfect repeats known as the microtubule binding repeats. Tau has recently been identified to have the ability to phase separate in vitro and in cells. The ability of tau to adopt a wide variety of conformations appears fundamental both to its biological function and also its association with neurodegenerative diseases. The recently highlighted involvement of low-complexity domains in liquid-liquid phase separation provides a critical link between the soluble function and the insoluble dysfunctional properties of tau.
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Project description:A significant part of the proteome is composed of intrinsically disordered proteins (IDPs). These proteins do not fold into a well-defined structure and behave like ordinary polymers. In this work, we consider IDPs that have the tendency to aggregate, model them as heteropolymers that contain a small number of associating monomers, and use computer simulations to compare the aggregation of such IDPs that are grafted to a surface or free in solution. We then discuss how such grafting may affect the analysis of in vitro experiments and could also be used to suppress harmful aggregation.
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Project description:The broad family of LEA proteins are intrinsically disordered proteins (IDPs) with several potential roles in desiccation tolerance, or anhydrobiosis, one of which is to limit desiccation-induced aggregation of cellular proteins. We show here that this activity, termed molecular shield function, is distinct from that of a classical molecular chaperone, such as HSP70 - while HSP70 reduces aggregation of citrate synthase (CS) on heating, two LEA proteins, a nematode group 3 protein, AavLEA1, and a plant group 1 protein, Em, do not; conversely, the LEA proteins reduce CS aggregation on desiccation, while HSP70 lacks this ability. There are also differences in interaction with client proteins - HSP70 can be co-immunoprecipitated with a polyglutamine-containing client, consistent with tight complex formation, whereas the LEA proteins can not, although a loose interaction is observed by Förster resonance energy transfer. In a further exploration of molecular shield function, we demonstrate that synthetic polysaccharides, like LEA proteins, are able to reduce desiccation-induced aggregation of a water-soluble proteome, consistent with a steric interference model of anti-aggregation activity. If molecular shields operate by reducing intermolecular cohesion rates, they should not protect against intramolecular protein damage. This was tested using the monomeric red fluorescent protein, mCherry, which does not undergo aggregation on drying, but the absorbance and emission spectra of its intrinsic fluorophore are dramatically reduced, indicative of intramolecular conformational changes. As expected, these changes are not prevented by AavLEA1, except for a slight protection at high molar ratios, and an AavLEA1-mCherry fusion protein is damaged to the same extent as mCherry alone. A recent hypothesis proposed that proteomes from desiccation-tolerant species contain a higher degree of disorder than intolerant examples, and that this might provide greater intrinsic stability, but a bioinformatics survey does not support this, since there are no significant differences in the degree of disorder between desiccation tolerant and intolerant species. It seems clear therefore that molecular shield function is largely an intermolecular activity implemented by specialist IDPs, distinct from molecular chaperones, but with a role in proteostasis.
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Project description:Intrinsically disordered proteins (IDPs) can be degraded in a ubiquitin-independent process by the 20S proteasome. Decline in 20S activity characterizes neurodegenerative diseases. Here, we examine 20S degradation of IDP tau, a protein that aggregates into insoluble deposits in Alzheimer's disease. We show that cleavage of tau by the 20S proteasome is most efficient within the aggregation-prone repeat region of tau and generates both short, aggregation-deficient peptides and two long fragments containing residues 1 to 251 and 1 to 218. Phosphorylation of tau by the non-proline-directed Ca2+/calmodulin-dependent protein kinase II inhibits degradation by the 20S proteasome. Phosphorylation of tau by GSK3β, a major proline-directed tau kinase, modulates tau degradation kinetics in a residue-specific manner. The study provides detailed insights into the degradation products of tau generated by the 20S proteasome, the residue specificity of degradation, single-residue degradation kinetics, and their regulation by posttranslational modification.
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Project description:The aggregation of the intrinsically disordered tau protein into highly ordered β-sheet-rich fibrils is implicated in the pathogenesis of a range of neurodegenerative disorders. The mechanism of tau fibrillogenesis remains unresolved, particularly early events that trigger the misfolding and assembly of the otherwise soluble and stable tau. We investigated the role the lipid membrane plays in modulating the aggregation of three tau variants, the largest isoform hTau40, the truncated construct K18, and a hyperphosphorylation-mimicking mutant hTau40/3Epi. Despite being charged and soluble, the tau proteins were also highly surface active and favorably interacted with anionic lipid monolayers at the air/water interface. Membrane binding of tau also led to the formation of a macroscopic, gelatinous layer at the air/water interface, possibly related to tau phase separation. At the molecular level, tau assembled into oligomers composed of ~ 40 proteins misfolded in a β-sheet conformation at the membrane surface, as detected by in situ synchrotron grazing-incidence X-ray diffraction. Concomitantly, membrane morphology and lipid packing became disrupted. Our findings support a general tau aggregation mechanism wherein tau's inherent surface activity and favorable interactions with anionic lipids drive tau-membrane association, inducing misfolding and self-assembly of the disordered tau into β-sheet-rich oligomers that subsequently seed fibrillation and deposition into diseased tissues.
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Project description:Sml1 is a small ribonucleotide reductase (RNR) regulatory protein in Saccharomyces cerevisiae that binds to and inhibits RNR activation. NMR studies of 15N-labeled Sml1 (104 residues), as well as of a truncated variant (residues 50-104), have allowed characterization of their molecular properties. Sml1 belongs to the class of intrinsically disordered proteins with a high degree of dynamics and very little stable structure. Earlier suggestions for a dimeric structure of Sml1 were confirmed, and from translation diffusion NMR measurements, a dimerization dissociation constant of 0.1 mM at 4 degreesC could be determined. The hydrodynamic radius for the monomeric form of Sml1 was determined to be 23.4 A, corresponding to a protein size between those of a globular protein and a coil. Formation of a dimer results in a hydrodynamic radius of 34.4 A. The observed chemical shifts showed in agreement with previous studies two segments with transient helical structure, residues 4-20 and 60-86, and relaxation studies clearly showed restricted motion in these segments. A spin-label attached to C14 showed long-range interactions with residues 60-70 and 85-95, suggesting that the N-terminal domain folds onto the C-terminal domain. Importantly, protease degradation studies combined with mass spectrometry indicated that the N-terminal domain is degraded before the C-terminal region and thus may serve as a protection against proteolysis of the functionally important C-terminal region. Dimer formation was not associated with significant induction of structure but was found to provide further protection against proteolysis. We propose that this molecular shielding and protection of vital functional structures from degradation by functionally unimportant sites may be a general attribute of other natively disordered proteins.
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