In Alzheimer disease, the microtubule-associated protein tau dissociates from the neuronal cytoskeleton and aggregates to form cytoplasmic inclusions. Although hyper-phosphorylation of tau Ser and Thr residues is an established trigger of tau misfunction and aggregation, tau modifications extend to Lys residues as well, raising the possibility that different modification signatures depress or promote aggregation propensity depending on site occupancy. To identify Lys-residue modifications associated with normal tau function, soluble tau proteins isolated from four cognitively normal human brains were characterized by mass spectrometry methods. The major detectable Lys modification was found to be methylation, which appeared in the form of mono- and di-methyl Lys residues distributed among at least eleven sites. Unlike tau phosphorylation sites, the frequency of Lys methylation was highest in the microtubule binding repeat region that mediates both microtubule binding and homotypic interactions. When purified recombinant human tau was modified in vitro through reductive methylation, its ability to promote tubulin polymerization was retained, whereas its aggregation propensity was greatly attenuated at both nucleation and extension steps. These data establish Lys methylation as part of the normal tau post-translational modification signature in human brain, and suggest that it can function in part to protect against pathological tau aggregation.
Since the discovery of phenothiazines as tau protein aggregation inhibitors, many additional small molecule inhibitors of diverse chemotype have been discovered and characterized in biological model systems. Although direct inhibition of tau aggregation has shown promise as a potential treatment strategy for depressing neurofibrillary lesion formation in Alzheimer’s disease, the mechanism of action of these compounds has been unclear. However, recent studies have found that tau aggregation antagonists exert their effects through both covalent and non-covalent means, and have identified associated potency and selectivity driving features. Here we review small-molecule tau aggregation inhibitors with a focus on compound structure and inhibitory mechanism. The elucidation of inhibitory mechanism has implications for maximizing on-target efficacy while minimizing off-target side effects.
Tau is a microtubule-associated protein that normally interacts in monomeric form with the neuronal cytoskeleton. In Alzheimer's disease, however, it aggregates to form the structural component of neurofibrillary lesions. The transformation is controlled in part by age-and diseaseassociated post-translational modifications. Recently we reported that tau isolated from cognitively normal human brain was methylated on lysine residues, and that high-stoichiometry methylation depressed tau aggregation propensity in vitro. However, whether methylation stoichiometry
Post-translational modifications are biologically important and wide-spread modulators of protein function. Although methods for detecting the presence of specific modifications are becoming established, approaches for quantifying their mol modification/mol protein stoichiometry are less well developed. Here we introduce a ratiometric, label-free, targeted liquid chromatography tandem mass spectroscopy-based method for estimating Lys and Arg methylation stoichiometry on post-translationally modified proteins. Methylated Lys and Arg were detected with limits of quantification at low fmol and with linearity extending from 20 to 5000 fmol. This level of sensitivity allowed estimation of methylation stoichiometry from microgram quantities of various proteins, including those derived from either recombinant or tissue sources. The method also disaggregated total methylation stoichiometry into its elementary mono-, di-, and tri-methylated residue components. In addition to being compatible with kinetic experiments of protein methylation, the approach will be especially useful for characterizing methylation states of proteins isolated from cells and tissues.
Small molecules that bind tau-bearing neurofibrillary lesions are being sought for premortem diagnosis, staging, and treatment of Alzheimer’s disease and other tauopathic neurodegenerative diseases. The utility of these agents will depend on both their binding affinity and binding site density (Bmax). Previously we identified polarizability as a descriptor of protein aggregate binding affinity. To examine its contribution to binding site density, we investigated the ability of two closely related benzothiazole derivatives ((E)-2-[[4-(dimethylamino)phenyl]azo]-6-methoxybenzothiazole) and ((E)-2-[2-[4-(dimethylamino)phenyl]ethenyl]-6-methoxybenzothiazole)) that differed in polarizability to displace probes of high (Thioflavin S) and low (radiolabeled (E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-methoxy)styrylbenzene; IMSB) density sites. Consistent with their site densities, Thioflavin S completely displaced radiolabeled IMSB, but IMSB was incapable of displacing Thioflavin S. Although both benzothiazoles displaced the low Bmax IMSB probe, only the highly polarizable analog displaced near saturating concentrations of the Thioflavin S probe. Quantum calculations showed that high polarizability reflected extensive pi-electron delocalization fostered by the presence of electron donating and accepting groups. These data suggest that electron delocalization promotes ligand binding at a subset of sites on tau aggregates that are present at high density, and that optimizing this aspect of ligand structure can yield tau-directed agents with superior diagnostic and therapeutic performance.
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