Regulation And Mechanism Of Human Tau Protein Misfolding

Alzheimer disease (AD) is an irreversible neurodegenerative disease and has posed a serious threat to human health. One pathological hallmark of AD is the progressive accumulation of misfolded hyperphosphorylated microtubule-associated protein Tau inside neurons, forming neurofibrillary tangles and leading to synaptic dysfunction and neuronal death. Another hallmark of AD is the progressive accumulation of the protein fragment amyloid-P (plaques) outside neurons.The physiological functions of human Tau are to promote microtubule assembly and stability, to modulate the transport of vesicles and organelles along microtubules and to regulate neuronal signaling pathways. Thus the characterization of factors regulating the fibrillization and pathological function of Tau protein is of great importance to clarify the etiology of AD and to assist in the establishment of medical treatment. Here we study the mechanism of human Tau misfolding from the points of protein primary structure and molecular chaperone both in vitro and in an inducible cell model.Fibril-forming motif is the core of the formation from native proteins to amyloid filaments. Although it has been previously demonstrated that two fibril-forming motifs 275VQIINK280 (PHF6*) and 306VQIVYK311 (PHF6) are essential and sufficient for the fibrillization of human Tau in vitro, we do not know whether such motifs are also important for abnormal aggregation of human Tau protein in vivo and thereby induce AD. Here we study sequence-dependent abnormal aggregation of human Tau fragment Tau244-372 in an inducible cell model. As evidenced by confocal laser scanning microscopy, Western blot, and immunogold electron microscopy, fibril-forming motifs are essential and sufficient for abnormal aggregation of Tau244-372 in SH-SY5Y neuroblastoma cells induced by Congo red:when its two fibril-forming segments PHF6 and PHF6* are deleted, Tau244-372 does lose not only its ability to to form fibrils in SH-SY5Y cells but also its ability to induce phosphorylation of endogenous human Tau in SH-SY5Y cells, and the replacement of PHF6 and PHF6* with an unrelated amyloidogenic sequence IFQINS from human lysozyme does rescue the phosphorylation-inducing and fibril-forming abilities of Tau244-372 in SH-SY5Y cells. By contrast, insertion of a non-fibril forming peptide GGGGGG does not drive the disabled Tau244-372 to induce phosphorylation of endogenous human Tau and to misfold in SH-SY5Y cells. Therefore, over-expressed human Tau fragment-induced phosphorylation of endogeneous human Tau is sequence-dependent in an inducible cell model. Furthermore, as revealed by quantum dots based probes combined with annexin V staining, annexin V-FITC apoptosis detection assay, and immunofluorescence, fibril-forming motifs are essential and sufficient for early apoptosis of living SH-SY5Y cells induced by abnormal aggregation of Tau244-372. Our results suggest that fibril-forming motifs could be the determinants of Tau protein tending to misfold in living cells, thereby inducing neuronal apoptosis and causing the initiation and development of AD. We thus suggest that sequence-dependent aggregation could be a key mechanism for the misfolding of many amyloidogenic proteins. We also claim a link between Tau aggregation and toxicity in the form of apoptosis.Protein disulfide isomerase (PDI) has a variety of biological functions. As both an enzyme and a molecular chaperone, PDI can inhibit the aggregation of human Tau in vitro, and co-localize with neurofibrillary tangles in pathological diagnosis of AD. Therefore, we are aimed to study the interaction and regulation mechanism of human PDI with human Tau both in vitro and in an inducible cell model. By using bioinformatics we have predicted that the key interaction sites of PDI with human Tau fragment Tau244-372 are mainly located in its two CGXC active sites of thioredoxin-like catalytic a-domain and a'-domain. By using point mutation and isothermal titration calorimetry (ITC), we have demonstrated that PDI binds to Taui44-372 and its pathogenic mutant ?K280 mainly through its 4 Cys residues in catalytic a-domain and a'-domain. Wild-type PDI binds to Tau monomer, forming a 1:1 complex with moderate, micromolar affinity at physiological pH and near physiological ionic strength. Both double mutant C53A/C56A in a-domain and double mutant C397A/C400A in a'-domain bind to Tau monomer, forming a 2:1 complexes with moderate, micromolar affinity. No binding reaction for Tau monomer with quadruple mutant C53A/C56A/C397A/C400A is detected by ITC, demonstrating that the four Cys residues within two CGXC motifs of PDI, Cys-53, Cys-56, Cys-397, and Cys-400, are key residues in the interaction of PDI with Tau protein. As revealed by thioflavin T binding assay and transmission electron microscopy, compared with wild-type PDI, no remarkable change in the inhibitory effect of its cysteine mutants on Tau fibrillization in vitro is observed. As evidenced by confocal laser scanning microscopy and Western blot, both wild-type PDI and its quadruple mutant can remarkably inhibit pathogenic phosphorylation and abnormal aggregation of human Tau and its pathogenic mutant ?K280 in SH-SY5Y cells induced by Congo red. Therefore, PDI inhibits the pathological pathway of human Tau independent of its two CGXC active sites, and is likely to function as a molecular chaperone in inhibiting Tau pathogenic phosphorylation and fibrillization which needs more evidence to confirm. Our findings reveal new insights into the regulation mechanism of Tau protein misfolding by PDI and will be helpful to the understanding of the role of PDI in the cellular quality control mechanisms.Information obtained from this study will lead to a better understanding of the regulation and mechanism of human Tau protein misfolding and should be useful in the development of new therapeutic reagents for AD.