A general framework to characterize inhibitors of calmodulin: Use of calmodulin inhibitors to study the interaction between calmodulin and its calmodulin binding domains

AbstractThe prominent role of Ca2+ in cell physiology is mediated by a whole set of proteins involved in Ca2+-signal generation, deciphering and arrest. Among these intracellular proteins, calmodulin (CaM) known as a prototypical calcium sensor, serves as a ubiquitous carrier of the intracellular calcium signal in all eukaryotic cell types. CaM is assumed to be involved in many diseases including Parkinson, Alzheimer, and rheumatoid arthritis. Defects in some of many reaction partners of CaM might be responsible for disease symptoms. Several classes of drugs bind to CaM with unwanted side effects rather than specific therapeutic use. Thus, it may be more promising to concentrate at searching for pharmacological interferences with the CaM target proteins, in order to find tools for dissecting and investigating CaM-regulatory and modulatory functions in cells.In the present study, we have established a screening assay based on fluorescence polarization (FP) to identify a diverse set of small molecules that disrupt the regulatory function of CaM. The FP-based CaM assay consists in the competition of two fluorescent probes and a library of chemical compounds for binding to CaM.Screening of about 5300 compounds (Strasbourg Academic Library) by displacement of the probe yielded 39 compounds in a first step, from which 6 were selected. Those 6 compounds were characterized by means of calorimetry studies and by competitive displacement of two fluorescent probes interacting with CaM. Moreover, those small molecules were tested for their capability to displace 8 different CaM binding domains from CaM. Our results show that these CaM/small molecules interactions are not functionally equivalent.The strategy that has been set up for CaM is a general model for the development and validation of other CaM interactors, to decipher their mode of action, or rationally design more specific CaM antagonists. Moreover, this strategy may be used for other protein binding assays intended to screen for molecules with preferred binding activity. This article is part of a Special Issue entitled: 12th European Symposium on Calcium

Zinc induces temperature-dependent reversible self-assembly of Tau

Tau is an intrinsically disordered microtubule-associated protein that is implicated in several neurodegenerative disorders called tauopathies. In these diseases, Tau is found in the form of intracellular inclusions that consist of aggregated paired helical filaments (PHFs) in neurons. Given the importance of this irreversible PHF formation in neurodegenerative disease, Tau aggregation has been extensively studied. Several different factors, such as mutations or post translational modifications, have been shown to influence the formation of late-stage non-reversible Tau aggregates. It was recently shown that zinc ions accelerated heparin-induced oligomerization of Tau constructs. Indeed, in vitro studies of PHFs have usually been performed in the presence of additional co-factors, such as heparin, in order to accelerate their formation. Using turbidimetry, we investigated the impact of zinc ions on Tau in the absence of heparin and found that zinc is able to induce a temperature-dependent reversible oligomerization of Tau. The obtained oligomers were not amyloid-like and dissociated instantly following zinc chelation or a temperature decrease. Finally, a combination of isothermal titration calorimetry and dynamic light scattering experiments showed zinc binding to a high-affinity binding site and three low-affinity sites on Tau, accompanied by a change in Tau folding. Altogether, our findings stress the importance of zinc in Tau oligomerization. This newly identified Zn-induced oligomerization mechanism may be a part of a pathway different of and concurrent to Tau aggregation cascade leading to PHF formation

The elusive tau molecular structures: can we translate the recent breakthroughs into new targets for intervention?

Insights into tau molecular structures have advanced significantly in recent years. This field has been the subject of recent breakthroughs, including the first cryo-electron microscopy structures of tau filaments from Alzheimer’s and Pick’s disease inclusions, as well as the structure of the repeat regions of tau bound to microtubules. Tau structure covers various species as the tau protein itself takes many forms. We will here address a range of studies that help to define the many facets of tau protein structures and how they translate into pathogenic forms. New results shed light on previous data that need now to be revisited in order to up-date our knowledge of tau molecular structure. Finally, we explore how these data can contribute the important medical aspects of this research - diagnosis and therapeutics

Cation binding to 15-TBA quadruplex DNA is a multiple-pathway cation-dependent process

A combination of explicit solvent molecular dynamics simulation (30 simulations reaching 4 µs in total), hybrid quantum mechanics/molecular mechanics approach and isothermal titration calorimetry was used to investigate the atomistic picture of ion binding to 15-mer thrombin-binding quadruplex DNA (G-DNA) aptamer. Binding of ions to G-DNA is complex multiple pathway process, which is strongly affected by the type of the cation. The individual ion-binding events are substantially modulated by the connecting loops of the aptamer, which play several roles. They stabilize the molecule during time periods when the bound ions are not present, they modulate the route of the ion into the stem and they also stabilize the internal ions by closing the gates through which the ions enter the quadruplex. Using our extensive simulations, we for the first time observed full spontaneous exchange of internal cation between quadruplex molecule and bulk solvent at atomistic resolution. The simulation suggests that expulsion of the internally bound ion is correlated with initial binding of the incoming ion. The incoming ion then readily replaces the bound ion while minimizing any destabilization of the solute molecule during the exchange

Bacterial co-expression of human Tau protein with protein kinase A and 14-3-3 for studies of 14-3-3/phospho-Tau interaction.

Abundant regulatory 14-3-3 proteins have an extremely wide interactome and coordinate multiple cellular events via interaction with specifically phosphorylated partner proteins. Notwithstanding the key role of 14-3-3/phosphotarget interactions in many physiological and pathological processes, they are dramatically underexplored. Here, we focused on the 14-3-3 interaction with human Tau protein associated with the development of several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Among many known phosphorylation sites within Tau, protein kinase A (PKA) phosphorylates several key residues of Tau and induces its tight interaction with 14-3-3 proteins. However, the stoichiometry and mechanism of 14-3-3 interaction with phosphorylated Tau (pTau) are not clearly elucidated. In this work, we describe a simple bacterial co-expression system aimed to facilitate biochemical and structural studies on the 14-3-3/pTau interaction. We show that dual co-expression of human fetal Tau with PKA in Escherichia coli results in multisite Tau phosphorylation including also naturally occurring sites which were not previously considered in the context of 14-3-3 binding. Tau protein co-expressed with PKA displays tight functional interaction with 14-3-3 isoforms of a different type. Upon triple co-expression with 14-3-3 and PKA, Tau protein could be co-purified with 14-3-3 and demonstrates complex which is similar to that formed in vitro between individual 14-3-3 and pTau obtained from dual co-expression. Although used in this study for the specific case of the previously known 14-3-3/pTau interaction, our co-expression system may be useful to study of other selected 14-3-3/phosphotarget interactions and for validations of 14-3-3 complexes identified by other methods

Deciphering the molecular mechanisms of anti-tubulin plant derived drugs

International audienceEven though commercialized anticancer drugs are now produced by pharmaceutical companies, most of them were originally obtained from natural sources, and more particularly from plants. Indeed, many structurally diverse compounds isolated from plants or marine flora have been purified and synthesized for their anticancer bioactivity. Among these, several molecules belong to the class of anticancer drugs which target the microtubule cytoskeleton, either by stabilizing it or destabilizing it. To characterize the activity of these drugs and to understand in which physiological context they are more likely to be used as therapeutic agents, it is necessary to fully determine their interaction with tubulin. Understanding the molecular basis of their effects on microtubule cytoskeleton is an important step in designing analogs with greater pharmacological activity and with fewer side effects. In addition, knowing the molecular mechanism of action of each drug that is already used in chemotherapy protocols will also help to find strategies to circumvent resistance. By taking examples of known anti-tubulin plant derived drugs, we show how identification of microtubule targeting agents and further characterization of their activity can be achieved combining biophysical and biochemical approaches. We also illustrate how continuing in depth study of molecules with already known primary mechanisms of action can lead to the discovery of new targets or biomarkers which can open newperspectives in anticancer strategies

Co-expression system proposed.

<p>Scheme demonstrates the pipelines in the case of the proposed dual (left side) or triple (right side) co-expression/purification system dealing with production of a phosphotarget or a 14-3-3/phosphotarget complex, respectively, using Tau protein as an example. Protein components of the system are coded by yellow rectangle (14-3-3), orange triangle (PKA), and green oval (Tau); light and dark oval stand for unphosphorylated and phosphorylated Tau, respectively. The His-tag on PKA and Tau is shown by a short blue curves. Note, that 3C protease was removed during IMAC2 because it contained an uncleavable His₆-tag. IMAC stands for immobilized metal-affinity chromatography.</p

Validation of interaction between pTau^coex and 14-3-3ζ by means of native gel-electrophoresis and analytical size-exclusion chromatography.

<p><b>A.</b> Purified 14-3-3ζ (lane 1) or its mixture with 1x (lane 2) or 2x (lane 3) amount of pTau^coex run on non-denaturing gel-electrophoresis in a 15% gel. 14-3-3ζ was present in equal amounts on lanes 1–3 but migrated to the intermediate (tentative complex) band in the presence of pTau which has pI>9 and does not enter the gel at pH 8.6. <b>B.</b> The tentative complex band was excised from the native gel and analyzed by SDS-PAGE to show the presence of both partners (lane 1). Lane 2, pTau^coex control; lane 3, 14-3-3ζ control; M, molecular mass standards (15, 25, 35, 40, 50, 70, 100, 140, 260 kDa). Arrows indicate positions of 14-3-3 and pTau. The gels were stained with freshly prepared Coomassie brilliant blue. <b>C.</b> Elution profiles of purified 14-3-3ζ, pTau^coex, their mixture, or algebraic sum of the individual profiles followed by absorbance at 227 nm using a Superdex200 Increase 10/300 column (GE Healthcare) operated at a flow rate of 1.2 ml/min. At least three experiments were performed with essentially similar results.</p

Components of the co-expression system.

<p>Components of the co-expression system.</p

Identification by in-gel trypsinolysis and tandem mass-spectrometry of phosphorylated sites within human fetal pTau^coex in <i>E</i>. <i>coli</i>.

<p>Numbering for two alternatively spliced Tau isoforms is shown. Likelihood that the identified phosphopeptide (underlined) serves as a 14-3-3-binding site is estimated from “-”(low probability) to “+++” (high probability), compared to the existing literature (right column). Sites of trypsinolysis is designated by “.”.</p