Phosphorylation Patterns, Aggregation Propensities, and Morphological Studies of The Various Tau Protein Isoforms

Tau protein is a microtubule-binding protein as well as a biomarker of neurodegeneration. Its core function is to stabilize microtubules for proper neuronal communication. When hyperphosphorylated, it detaches itself from microtubules and self-assembles into cytotoxic structures. However, little is known about how phosphorylation, the commonest posttranslational modification process found in eukaryotic cells regulate Tau protein structure, conformation, and function. Herein, the role of specific kinases or kinase combinations from the three main classes of proteins kinases that phosphorylate Tau: Proline-directing protein kinase (Glycogen synthase kinase (GSK-3β)) and non-proline directing protein kinase (Microtubule associated regulating kinase (MARK4)) and Tyrosine kinase (Fyn) were systematically evaluated in vitro. After the expression and purification of all the six Tau isoforms from E. Coli cells, Tau 441, also known as full-length Tau, which comprises of all the domains found in the other isoforms was extensively investigated. Phosphorylation of Tau 441 by GSK-3β, MARK4 and Fyn was detected by immunostaining using phosphospecific antibodies. With Tau protein been identified to co-localize with sulfated aminoglycans such as heparin and heparin sulfates, single and multi-kinase phosphorylated aggregation studies of Tau 441 were conducted in the presence or absence of heparin. Functional assays including proteostat assays, turbidity assays and SDS-PAGE were used to evaluate the aggregation properties of Tau 441 after phosphorylation.The phosphosites on all the single and multi-kinase phosphorylated Tau 441 samples were characterized by Tandem mass spectrometry. Tau 441 protein structure and conformational changes after phosphorylation was also determined by Hydrogen Deuterium Exchange mass spectrometry (HDX-MS). The flexibility and accessibility to the first and second hexapeptide repeats (H1 and H2), a repeat motif found in the MTBR of Tau protein identified to increase the aggregation tendencies of Tau protein were used to describe the single kinase or multi-kinase combinations evaluated ability to promote Tau fibrillization.