Site‐Specific Hyperphosphorylation Inhibits, Rather than Promotes, Tau Fibrillization, Seeding Capacity, and Its Microtubule Binding

Abstract

The consistent observation of phosphorylated tau in the pathology of Alzheimer's disease has contributed to the emergence of a model where hyperphosphorylation triggers both tau disassociation from microtubules and its subsequent aggregation. Herein, we applied a total chemical synthetic approach to site‐specifically phosphorylate the microtubule binding repeat domain of tau (K18) at single (pS356) or multiple (pS356/pS262 and pS356/pS262/pS258) residues. We show that hyperphosphorylation of K18 inhibits 1) its aggregation in vitro, 2) its seeding activity in cells, 3) its binding to microtubules, and 4) its ability to promote microtubule polymerization. The inhibition increased with increasing the number of phosphorylated sites, with phosphorylation at S262 having the strongest effect. Our results argue against the hyperphosphorylation hypothesis and underscore the importance of revisiting the role of site‐specific hyperphosphorylation in regulating tau functions in health and disease.

Semisynthetic Modification of Tau Protein with Di-Ubiquitin Chains for Aggregation Studies

“…This effect was found to be independent of the di-ubiquitin linkage topology ( Figure 5 d). Our findings are consistent with previous studies reporting that multiple mono-ubiquitination of tau protein reduces MT binding affinity [ 43 ], and phosphorylation at Ser356 (close to Lys353) slightly impairs MT assembly [ 37 ]. Taken together, the evidence on site-specific phosphorylated or on multi mono-ubiquitinated tau protein, and our results obtained with the di-ubiquitinated proteins indicate that post-translational modifications around position 353 do not crucially affect tau-assisted MT polymerization and that the functional activity of tau protein is highly dependent on the sites of modification.…”

“…Microtubule (MT) polymerization was monitored by following the increase in absorbance at 350 nm. The kinetics of MT assembly in the presence of tau4RDΔC resembled previous results reported on tau4RD [ 37 ]. The data acquired in the presence of Ub 2 (48)tau4RD(353) or Ub 2 (63)tau4RD(353) indicates that the presence of the di-ubiquitin chains at position 353 of tau protein, moderately but significantly inhibits MT polymerization.…”

“…To interpret our results in the wider context of post-translational modifications of tau protein, we reviewed recent studies which explored the impact of single or multiple phosphorylation of full-length tau protein or tau4RD [ 37 , 41 ]. Phosphorylation of both Ser262 and Ser356 was found to significantly alter the aggregation kinetics of tau4RD [ 37 ], and single-site phosphorylation affected tau protein aggregation in a sequence-specific manner [ 37 , 41 ]: phosphorylation of Ser305 had a significant impact on fibril formation; however modification at Ser356 did not significantly perturb the aggregation of tau protein. Here we have shown that di-ubiquitination at 353, close to residue 356, allows the formation of fibrils, although with a reduced aggregation rate, in line with the observation that this site is excluded from the ordered core (residues 272–330) in heparin-induced tau filaments [ 42 ].…”

Phosphorylation of the overlooked tyrosine 310 regulates the structure, aggregation, and microtubule- and lipid-binding properties of Tau

“…Despite consistent findings implicating PHF6 in the initiation of Tau aggregation, stabilization of Tau fibrils, and the importance of the interaction of Tyr-310 and Ile-308 for Tau filament formation (35), there are no reports in the literature examining the potential impact of phosphorylation at this residue on the physiological and/or pathogenic properties of biologically relevant fulllength Tau proteoforms. This is possibly due to the lack of wellcharacterized pTyr-310 -specific antibodies and until recently the lack of methods for site-specific phosphorylation of Tau (57)(58)(59).…”

Introductory Chapter: Phosphorus - Nature’s Versatile Pentavalent and Tetrahedral Covalent Building Block and Agent for Energy, Disease and Health

“…ATP is an energy storage molecule for life; related molecules also possess the same energy in the anhydride P-O-P bonds; as seen in Table 1, considering the number of hits (articles), and looking a little deeper suggesting there may be a lot of research momentum in the field. Furthermore, post-translational modifications (PTM's), for example in the form of phosphorylation, are exceptionally interesting and important; research with this chemistry continues ( Table 2); the Park laboratory at KAIST for example, has found ways to explore phosphoserine "stop codon" chemistry and to apply this biosynthesis to various problems in molecular biology [13][14][15][16]; Additionally, the Lashuel group at EPFL and other researchers (e.g., E. Mandelkow) have been involved with detailed studies of phosphorylation, specifically in the context of neurodegenerative disease research [17][18][19][20]. The presence of a phosphorylated serine, threonine, tyrosine, etc., reveals the evident workings of kinases and anticipation of the workings of phosphatases.…”