RESUMEN
Abnormal accumulation of tau proteins is one pathological hallmark of Alzheimerâ¡s disease (AD). Many tau protein post-translational modifications (PTMs) are associated with the development of AD, such as phosphorylation, acetylation, and methylation. Therefore, a complete picture of PTM landscape of tau is critical for understanding the molecular mechanisms of AD progression. Here, we offered a pilot study of combining two complementary analytical techniques, capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) and reversed-phase liquid chromatography (RPLC)-MS/MS, for bottom-up proteomics of recombinant human tau-0N3R. We identified 53 phosphorylation sites of tau-0N3R in total, which is about 30% higher than that from RPLC-MS/MS alone. CZE-MS/MS provided more PTM sites (i.e., phosphorylation) and modified peptides of tau-0N3R than RPLC-MS/MS, and its predicted electrophoretic mobility helped improve the confidence of the identified modified peptides. We developed a highly efficient capillary isoelectric focusing (cIEF)-MS technique to offer a bird's-eye view of tau-0N3R proteoforms, with 11 putative tau-0N3R proteoforms carrying up to nine phosphorylation sites and lower pI values from more phosphorylated proteoforms detected. Interestingly, under a native-like cIEF-MS condition, we observed three putative tau-0N3R dimers carrying phosphate groups. The findings demonstrate that CE-MS is a valuable analytical technique for the characterization of tau PTMs, proteoforms, and even oligomerization.
RESUMEN
Abnormal phosphorylation of the microtubule-binding protein tau in the brain is a key pathological marker for Alzheimer's disease and additional neurodegenerative tauopathies. However, how hyperphosphorylated tau causes cellular dysfunction or death that underlies neurodegeneration remains an unsolved question critical for the understanding of disease mechanism and the design of efficacious drugs. Using a recombinant hyperphosphorylated tau protein (p-tau) synthesized by the PIMAX approach, we examined how cells responded to the cytotoxic tau and explored means to enhance cellular resistance to tau attack. Upon p-tau uptake, the intracellular calcium levels rose promptly. Gene expression analyses revealed that p-tau potently triggered endoplasmic reticulum (ER) stress, unfolded protein response (UPR), ER stress-associated apoptosis, and pro-inflammation in cells. Proteomics studies showed that p-tau diminished heme oxygenase-1 (HO-1), an ER stress-associated anti-inflammation and anti-oxidative stress regulator, while stimulated the accumulation of MIOS and other proteins. p-Tau-induced ER stress-associated apoptosis and pro-inflammation are ameliorated by apomorphine, a brain-permeable prescription drug widely used to treat Parkinson's disease symptoms, and by overexpression of HO-1. Our results reveal probable cellular functions targeted by hyperphosphorylated tau. Some of these dysfunctions and stress responses have been linked to neurodegeneration in Alzheimer's disease. The observations that the ill effects of p-tau can be mitigated by a small compound and by overexpressing HO-1 that is otherwise diminished in the treated cells inform new directions of Alzheimer's disease drug discovery.
RESUMEN
Background: Abnormal phosphorylation of the microtubule-binding protein tau in the brain is a key pathological marker for Alzheimer's disease and additional neurodegenerative tauopathies. However, how hyperphosphorylated tau causes cellular dysfunction or death that underlie neurodegeneration remains an unsolved question critical for the understanding of disease mechanism and the design of efficacious drugs. Methods: Using a recombinant hyperphosphorylated tau protein (p-tau) synthesized by the PIMAX approach, we examined how cells responded to the cytotoxic tau and explored means to enhance cellular resistance to tau attack. Results: Upon p-tau uptake, the intracellular calcium levels rose promptly. Gene expression analyses revealed that p-tau potently triggered endoplasmic reticulum (ER) stress, Unfolded Protein Response (UPR), ER stress-associated apoptosis, and pro-inflammation in cells. Proteomics studies showed that p-tau diminished heme oxygenase-1 (HO-1), an ER stress associated anti-inflammation and anti-oxidative stress regulator, while stimulated the accumulation of MIOS and other proteins. P-tau-induced ER stress-associated apoptosis and pro-inflammation are ameliorated by apomorphine, a brain-permeable prescription drug widely used to treat Parkinson's disease symptoms, and by overexpression of HO-1. Conclusion: Our results reveal probable cellular functions targeted by hyperphosphorylated tau. Some of these dysfunctions and stress responses have been linked to neurodegeneration in Alzheimer's disease. The observations that the ill effects of p-tau can be mitigated by a small compound and by overexpressing HO-1 that is otherwise diminished in the treated cells inform new directions of Alzheimer's disease drug discovery.