NMR spectroscopy of the neuronal tau protein: normal function and implication in Alzheimer's disease

Abstract

NMR spectroscopy was used to explore the different aspects of the normal and pathological functions of tau, but proved challenging because the protein contains 441 amino acids and has poor signal dispersion. We have set out to dissect the phosphorylation patterns of tau in order to understand better its role in the aggregation process and microtubule-binding regulation. Our current knowledge on the functional consequences of specific phosphorylations is still limited, mainly because producing and assessing quantitatively phosphorylated tau samples is far from straightforward, even in vitro. We use NMR spectroscopy as a proteomics tool to characterize the phosphorylation patterns of tau, after in vitro phosphorylation by recombinant kinases. The phosphorylated tau can next be use for functional assays or interaction assays with phospho-dependent protein partners, such as the prolyl cis-trans isomerase Pin1.

Post‐translational modification: nature's escape from genetic imprisonment and the basis for dynamic information encoding

“…Competition between serine/threonine phosphorylation and GlcNAcylation, via the “yin-yang” mechanism (107), has been suggested as a potential therapeutic strategy against Alzheimer’s (119). Determining the corresponding mod-form distributions may offer key insights into this devastating disease (64). …”

“…Nuclear magnetic resonance spectroscopy is a complementary method, that is more limited in scope than MS but has real-time and even in-vivo potential. Studies of tau phosphoryl-forms by NMR, undertaken by one of our labs, suggest what can be achieved for a large unstructured protein with complex phosphorylation patterns (64). …”

Physiological and pathological phosphorylation of tau by Cdk5

“…Liu et al (2002) reported that Cdk5-p25 phosphorylates Tau at Thr181, Ser199, Ser202, Thr205, Thr212, Ser214, Ser217, Thr231, Ser235, Ser396, and Ser404 using a repertoire of phospho-specific antibodies. Using NMR, Landrieu et al (2010, 2011) analyze tau phosphorylation by Cdk2-cyclin A3 and Cdk5-p25; while Cdk2-cyclin A3 phosphorylated Thr153, Ser199, Ser202, Thr205, Thr231, Ser235, and Ser404, with high levels of phosphate incorporation at Ser202/Thr205 and Thr231/Ser235, Cdk5-p25 needed GSK3β for the same phosphorylation profile with Cdk5-p25 providing Ser202, Thr205, Ser235, and Ser404 as major sites. We believe that these differences in phosphorylation sites were due to the methods and kinase preparations used for analysis.…”

The Prolyl Isomerase Pin1 Targets Stem-Loop Binding Protein (SLBP) To Dissociate the SLBP-Histone mRNA Complex Linking Histone mRNA Decay with SLBP Ubiquitination

“…Since the interaction of Pin1 with its substrates has been well characterized by NMR (13,19,(26)(27)(28)43) and X-ray crystallography (45,54,66), this approach allowed us to determine whether SLBP interacted with Pin1 in a specific manner. In this assay, phosphorylated SLBP at natural isotope abundance was titrated into a solution of "NMR active"…”