Nejvíce citovaný článek - PubMed ID 30884818
Structure and Functions of Microtubule Associated Proteins Tau and MAP2c: Similarities and Differences
Microtubule-associated protein 2 (MAP2) is an important neuronal target of extracellular signal-regulated kinase 2 (ERK2) involved in Raf signaling pathways, but mechanistic details of MAP2 phosphorylation are unclear. Here, we used NMR spectroscopy to quantitatively describe the kinetics of phosphorylation of individual serines and threonines in the embryonic MAP2 variant MAP2c. We carried out real-time monitoring of phosphorylation to discover major phosphorylation sites that were not identified in previous studies relying on specific antibodies. Our comparison with the phosphorylation of MAP2c by a model cyclin-dependent kinase CDK2 and with phosphorylation of the MAP2c homolog Tau revealed differences in phosphorylation profiles that explain specificity of regulation of biological functions of MAP2c and Tau. To probe the molecular basis of the regulatory effect of ERK2, we investigated the interactions of phosphorylated and unphosphorylated MAP2c by NMR with single-residue resolution. As ERK2 phosphorylates mostly outside the regions binding microtubules, we studied the binding of proteins other than tubulin, namely regulatory subunit RIIα of cAMP-dependent PKA, adapter protein Grb2, Src homology domain 3 of tyrosine kinases Fyn and Abl, and ERK2 itself. We found ERK2 phosphorylation interfered mostly with binding to proline-rich regions of MAP2c. Furthermore, our NMR experiments in SH-SY5Y neuroblastoma cell lysates showed that the kinetics of dephosphorylation are compatible with in-cell NMR studies and that residues targeted by ERK2 and PKA are efficiently phosphorylated in the cell lysates. Taken together, our results provide a deeper characterization of MAP2c phosphorylation and its effects on interactions with other proteins.
- Klíčová slova
- NMR, PKA, Src homology 3 domain, cyclin-dependent kinase, extracellular signal–regulated kinase, growth factor receptor-bound protein 2 (GRB2), microtubule-associated protein,
- MeSH
- extracelulárním signálem regulované MAP kinasy * metabolismus MeSH
- fosforylace MeSH
- lidé MeSH
- mikrotubuly metabolismus MeSH
- nádorové buněčné linie MeSH
- proteinkinasy řízené prolinem * metabolismus MeSH
- proteiny asociované s mikrotubuly * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- CDK2 protein, human MeSH Prohlížeč
- extracelulárním signálem regulované MAP kinasy * MeSH
- MAP2 protein, human MeSH Prohlížeč
- MAPK1 protein, human MeSH Prohlížeč
- proteinkinasy řízené prolinem * MeSH
- proteiny asociované s mikrotubuly * MeSH
Tauopathies, including Alzheimer's disease (AD), are the most troublesome of all age-related chronic conditions, as there are no well-established disease-modifying therapies for their prevention and treatment. Spatio-temporal distribution of tau protein pathology correlates with cognitive decline and severity of the disease, therefore, tau protein has become an appealing target for therapy. Current knowledge of the pathological effects and significance of specific species in the tau aggregation pathway is incomplete although more and more structural and mechanistic insights are being gained using biophysical techniques. Here, we review the application of NMR to structural studies of various tau forms that appear in its aggregation process, focusing on results obtained from solid-state NMR. Furthermore, we discuss implications from these studies and their prospective contribution to the development of new tauopathy therapies.
- Klíčová slova
- Alzheimer’s disease, filaments, nuclear magnetic resonance, protein structure, tau,
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Drug withdrawal is associated with abstinence symptoms including deficits in cognitive functions that may persist even after prolonged discontinuation of drug intake. Cognitive deficits are, at least partially, caused by alterations in synaptic plasticity but the precise molecular mechanisms have not yet been fully identified. In the present study, changes in proteomic and phosphoproteomic profiles of selected brain regions (cortex, hippocampus, striatum, and cerebellum) from rats abstaining for six months after cessation of chronic treatment with morphine were determined by label-free quantitative (LFQ) proteomic analysis. Interestingly, prolonged morphine withdrawal was found to be associated especially with alterations in protein phosphorylation and to a lesser extent in protein expression. Gene ontology (GO) term analysis revealed enrichment in biological processes related to synaptic plasticity, cytoskeleton organization, and GTPase activity. More specifically, significant changes were observed in proteins localized in synaptic vesicles (e.g., synapsin-1, SV2a, Rab3a), in the active zone of the presynaptic nerve terminal (e.g., Bassoon, Piccolo, Rims1), and in the postsynaptic density (e.g., cadherin 13, catenins, Arhgap35, Shank3, Arhgef7). Other differentially phosphorylated proteins were associated with microtubule dynamics (microtubule-associated proteins, Tppp, collapsin response mediator proteins) and the actin-spectrin network (e.g., spectrins, adducins, band 4.1-like protein 1). Taken together, a six-month morphine withdrawal was manifested by significant alterations in the phosphorylation of synaptic proteins. The altered phosphorylation patterns modulating the function of synaptic proteins may contribute to long-term neuroadaptations induced by drug use and withdrawal.
- Klíčová slova
- brain, morphine, proteomics, synaptic plasticity, withdrawal,
- Publikační typ
- časopisecké články MeSH
Biomolecular force fields optimized for globular proteins fail to properly reproduce properties of intrinsically disordered proteins. In particular, parameters of the water model need to be modified to improve applicability of the force fields to both ordered and disordered proteins. Here, we compared performance of force fields recommended for intrinsically disordered proteins in molecular dynamics simulations of three proteins differing in the content of ordered and disordered regions (two proteins consisting of a well-structured domain and of a disordered region with and without a transient helical motif and one disordered protein containing a region of increased helical propensity). The obtained molecular dynamics trajectories were used to predict measurable parameters, including radii of gyration of the proteins and chemical shifts, residual dipolar couplings, paramagnetic relaxation enhancement, and NMR relaxation data of their individual residues. The predicted quantities were compared with experimental data obtained within this study or published previously. The results showed that the NMR relaxation parameters, rarely used for benchmarking, are particularly sensitive to the choice of force-field parameters, especially those defining the water model. Interestingly, the TIP3P water model, leading to an artificial structural collapse, also resulted in unrealistic relaxation properties. The TIP4P-D water model, combined with three biomolecular force-field parameters for the protein part, significantly improved reliability of the simulations. Additional analysis revealed only one particular force field capable of retaining the transient helical motif observed in NMR experiments. The benchmarking protocol used in our study, being more sensitive to imperfections than the commonly used tests, is well suited to evaluate the performance of newly developed force fields.
