Characterization of the MAPT mutations Q336H and DelK280 using a new intramolecular FRET biosensor

Tau is a microtubule-associated protein mainly expressed in neurons, encoded by the MAPT gene. Mutations in the MAPT locus lead to abnormal accumulation of hyperphosporylated tau in abundant intracellular inclusions known as neurofibrillary tangles (NFTs), a common feature of a group of neurodegenerative diseases known as Tauopathies. In order to characterize the effect of ΔK280 and Q336H MAPT mutations FRET and FRAP techniques have been employed.To this aim the Conformational-Sensitive Tau Sensor (CST) that is based on the full length tau isoform 4R0N with the ECFP fused at the N-terminus and the EYFP at the C-terminus have been used. Since the CST allows to evaluate the changing in protein conformation and tau interactions with microtubules in living cells,two mutated CST constructs have been generated. The study has revealed that Q336H and ΔK280 induced conformational changes in tau protein.In particular, Q336H alters the conformation by allowing the two ends of Tau protein to approach each other in a more closed loop-like structure. On the contrary, ΔK280 determined a conformational change by inducing a more relaxed three-dimensional structure and altered Tau stability on Microtubules by increasing the soluble Tau protein

The Q336H MAPT mutation linked to Pick's disease leads to increased binding of tau to the microtubule network via altered conformational and phosphorylation effects

Tauopathies are neurodegenerative disorders characterized by Tau aggregation. Genetic studies on familial cases allowed for the discovery of mutations in the MAPT gene that increase Tau propensity to detach from microtubules and to form insoluble cytoplasmic Tau aggregates. Recently, the rare mutation Q336H has been identified to be associated with Pick's disease (PiD) and biochemical analyses demonstrated its ability to increase the microtubules (MTs) polymerization, thus revealing an opposite character compared to other Tau mutations studied so far. Here we investigated the biophysical and molecular properties of Tau(Q336H) in living cells by the employment of the conformational Tau biosensor CST. We found that this mutation alters Tau conformation on microtubules, stabilizes its binding to tubulin, and is associated with a paradoxical lower level of Tau phosphorylation. Moreover, we found that this mutation impacts the cytoskeletal complexity by increasing the tubulin filament length and the number of branches. However, despite these apparently non-pathological traits, we observed the formation of intracellular inclusions confirming that Q336H leads to aggregation. Our results suggest that the Tau aggregation process might be triggered by molecular mechanisms other than Tau destabilization or post-translational modifications which are likely to be detrimental to neuronal function in vivo

Screening Approaches for Targeting Ribonucleoprotein Complexes: A New Dimension for Drug Discovery.

RNA-binding proteins (RBPs) are pleiotropic factors that control the processing and functional compartmentalization of transcripts by binding primarily to mRNA untranslated regions (UTRs). The competitive and/or cooperative interplay between RBPs and an array of coding and noncoding RNAs (ncRNAs) determines the posttranscriptional control of gene expression, influencing protein production. Recently, a variety of well-recognized and noncanonical RBP domains have been revealed by modern system-wide analyses, underlying an evolving classification of ribonucleoproteins (RNPs) and their importance in governing physiological RNA metabolism. The possibility of targeting selected RNA-protein interactions with small molecules is now expanding the concept of protein "druggability," with new implications for medicinal chemistry and for a deeper characterization of the mechanism of action of bioactive compounds. Here, taking SF3B1, HuR, LIN28, and Musashi proteins as paradigmatic case studies, we review the strategies applied for targeting RBPs, with emphasis on the technological advancements to study protein-RNA interactions and on the requirements of appropriate validation strategies to parallel high-throughput screening (HTS) efforts

HuR modulation counteracts lipopolysaccharide response in murine macrophages

: Lipopolysaccharide (LPS) exposure to macrophages induces an inflammatory response, which is regulated at the transcriptional and post-transcriptional levels. HuR (ELAVL1) is an RNA-binding protein that regulates cytokines and chemokines transcripts containing AU/U-rich elements (AREs) and mediates the LPS-induced response. Here, we show that small-molecule tanshinone mimics (TMs) inhibiting HuR-RNA interaction counteract LPS stimulus in macrophages. TMs exist in solution in keto-enolic tautomerism, and molecular dynamic calculations showed the ortho-quinone form inhibiting binding of HuR to mRNA targets. TM activity was lost in vitro by blocking the diphenolic reduced form as a diacetate, but resulted in prodrug-like activity in vivo. RNA and ribonucleoprotein immunoprecipitation sequencing revealed that LPS induces a strong coupling between differentially expressed genes and HuR-bound genes, and TMs reduced such interactions. TMs decreased the association of HuR with genes involved in chemotaxis and immune response, including Cxcl10, Il1b and Cd40, reducing their expression and protein secretion in primary murine bone marrow-derived macrophages and in an LPS-induced peritonitis model. Overall, TMs show anti-inflammatory properties in vivo and suggest HuR as a potential therapeutic target for inflammation-related diseases