Plant calmodulin-like proteins: calcium binding and target interactions

Calcium (Ca2+) is an ubiquitous key second messenger in all living organisms, from bacteria to differentiated neuronal cells and is involved in a large variety of biological processes such as apoptosis, cell differentiation and proliferation, fertilization, transcription factors activation and many others. Plants in particular are known to be capable of exploiting Ca2+ signalling in a more sophisticated way than other organisms as demonstrated by the larger set of Ca2+-binding proteins (CBPs). Although the study of CBP has been an open and highly studied field for over a century, a biochemical and structural characterization of many CBPs from different organisms is still missing. Here, we provided a deep characterisation of two plant (Arabidopsis thaliana) Ca2+ sensors, a specific class of CBPs, to understand the differences in their ability to respond to Ca2+ and the regulation of downstream targets. Thus we analysed the metal and target binding properties of Calmodulin like protein 7 and 19 (CML7 and CML19). By exploiting spectroscopic techniques (NMR and CD) we evaluated the conformational changes induced by metal binding and assigned the specific affinities for each binding site by a combination of calorimetric and spectroscopic techniques (ITC and NMR). To further understand the Ca2+ sensing properties and the ability to interact with target proteins, we analysed the interaction of CML7 and CML19 with synthetic peptides, representing the binding region on physiological or model targets. Because the majority of CBPs are characterised by multiple metal binding sites, clustered affinities and cooperativity events, the determination of Ca2+/CBPs stoichiometry requires time and sample consuming procedures and sophisticated instrumentations (i.e. ITC and NMR). Thus, we report the optimisation of a new method, based on X-ray fluorescence, to overcome the most common limitations of the currently used techniques, allowing the quick estimation of the Ca2+/protein stoichiometry using low amounts of sample. Finally, initial studies on the recombinant production and characterisation of a neuronal family of CBPs are reported. These protein are called NECABs and are characterised by a peculiar domain composition and might be involved in the development of neurodegenerative diseases

Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs

Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 \u3bcM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 \u3bcM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability

Facile Cellulase Immobilisation on Bioinspired Silica

Cellulases are enzymes with great potential for converting biomass to biofuels for sustainable energy. However, their commercial use is limited by their costs and low reusability. Therefore, the scientific and industrial sectors are focusing on finding better strategies to reuse enzymes and improve their performance. In this work, cellulase from Aspergillus niger was immobilised through in situ entrapment and adsorption on bio-inspired silica (BIS) supports. To the best of our knowledge, this green effect strategy has never been applied for cellulase into BIS. In situ entrapment was performed during support synthesis, applying a one-pot approach at mild conditions (room temperature, pH 7, and water solvent), while adsorption was performed after support formation. The loading efficiency was investigated on different immobilisation systems by Bradford assay and FTIR. Bovine serum albumin (BSA) was chosen as a control to optimize cellulase loading. The residual activity of cellulase was analysed by the dinitro salicylic acid (DNS) method. Activity of 90% was observed for the entrapped enzyme, while activity of ~55% was observed for the adsorbed enzyme. Moreover, the supported enzyme systems were recycled five times to evaluate their reuse potential. The thermal and pH stability tests suggested that both entrapment and adsorption strategies can increase enzyme activity. The results highlight that the entrapment in BIS is a potentially useful strategy to easily immobilise enzymes, while preserving their stability and recycle potential

A Method for Metal/Protein Stoichiometry Determination Using Thin-Film Energy Dispersive X-ray Fluorescence Spectroscopy

A convenient approach is proposed for the quantitation of elemental cofactors in proteins and the determination of metal/protein stoichiometry, on the basis of energy dispersive X-ray fluorescence spectroscopy (EDXRF). The analysis of proteins containing the metals Cu, Fe, Zn, and Ca and also the nonmetallic element P is shown as a demonstration of the generality of the method. In general, the reported method gives limit of detection (LOD) and limit of quantification (LOQ) values in the low ppm range and requires only a few microliters of protein sample at micromolar concentrations. Moreover, sample preparation does not require any digestion steps before the analysis. The expected metal/protein stoichiometry was observed for each protein analyzed, highlighting the precision and accuracy of the method in all the tested cases. Furthermore, it is shown that the method is compatible with multimeric proteins and those with post-translational modifications such as glycosylation

Cation and peptide binding properties of CML7, a calmodulin-like protein from Arabidopsis thaliana

Plants contain a large family of so-called calmodulin-like proteins (CMLs) which differ from canonical calmodulin in that they show greater variability in sequence, length, and number of EF-hand domains. The presence of this extended CML family has raised questions regarding the role of these proteins: are they functionally redundant or do they play specific functions in physiological plant processes? To answer these questions, comprehensive biochemical and structural information on CML proteins is fundamental. Among the 50 CMLs from Arabidopsis thaliana, herein we described the ability of CML7 to bind metal ions focusing on the Ca2+ and Mg2+ sensing properties, as well as on metal-induced conformational changes. Circular dichroism and nuclear magnetic resonance (NMR) studies indicated that both Ca2+ and Mg2+ stabilize CML7, as reflected in conformational rearrangements in secondary and tertiary structure and in increases in thermal stability of the protein. However, the conformational changes that binding induces differ between the two metal ions, and only Ca2+ binding controls a structural transition that leads to hydrophobic exposure, as suggested by 8-anilino-1-naphthalenesulfonic acid fluorescence. Isothermal titration calorimetry data coupled with NMR experiments revealed the presence of two high affinity Ca2+-binding sites in the C-lobe of CML7 and two weaker sites in the N-lobe. The paired nature of these CML7 EF-hands enables them to bind Ca2+ with positive cooperativity within each globular domain. Our results clearly place CML7 in the category of Ca2+ sensors. Along with this, the protein can bind to a model target peptide (melittin) in a Ca2+-dependent manner

Binding of calcium and target peptide to calmodulin-like protein CML19, the centrin 2 of Arabidopsis thaliana

Calmodulin-like protein 19 (CML19) is an Arabidopsis centrin that modulates nucleotide excision repair (NER) by binding to RAD4 protein, the Arabidopsis homolog of human Xeroderma pigmentosum complementation group C protein. Although the necessity of CML19 as a part of the RAD4 plant recognition complex for functional NER is known at a cellular level, little is known at a molecular level. Herein, we used a combination of biophysical and biochemical approaches to investigate the structural and ion and target-peptide binding properties of CML19. We found that CML19 possesses four Ca2+-specific binding sites, two of high affinity in the N-terminal domain and two of low affinity in the C-terminal domain. Binding of Ca2+ to CML19 increases its alpha-helix content, stabilizes the tertiary structure, and triggers a conformational change, resulting in the exposure of a hydrophobic patch instrumental for target protein recognition. Using bioinformatics tools we identified a CML19-binding site at the C-terminus of RAD4, and through in vitro binding experiments we analyzed the interaction between a 17-mer peptide representing this site and CML19. We found that the peptide shows a high affinity for CML19 in the presence of Ca2+ (stoichiometry 1:1) and the interaction primarily involves the C-terminal half of CML19

Cation and peptide binding properties of CML7, a calmodulin-like protein from Arabidopsis thaliana

Plants contain a large family of so-called calmodulin-like proteins (CMLs) which differ from canonical calmodulin in that they show greater variability in sequence, length, and number of EF-hand domains. The presence of this extended CML family has raised questions regarding the role of these proteins: are they functionally redundant or do they play specific functions in physiological plant processes? To answer these questions, comprehensive biochemical and structural information on CML proteins is fundamental. Among the 50 CMLs from Arabidopsis thaliana, herein we described the ability of CML7 to bind metal ions focusing on the Ca2+ and Mg2+ sensing properties, as well as on metal-induced conformational changes. Circular dichroism and nuclear magnetic resonance (NMR) studies indicated that both Ca2+ and Mg2+ stabilize CML7, as reflected in conformational rearrangements in secondary and tertiary structure and in increases in thermal stability of the protein. However, the conformational changes that binding induces differ between the two metal ions, and only Ca2+ binding controls a structural transition that leads to hydrophobic exposure, as suggested by 8-anilino-1-naphthalenesulfonic acid fluorescence. Isothermal titration calorimetry data coupled with NMR experiments revealed the presence of two high affinity Ca2+-binding sites in the C-lobe of CML7 and two weaker sites in the N-lobe. The paired nature of these CML7 EF-hands enables them to bind Ca2+ with positive cooperativity within each globular domain. Our results clearly place CML7 in the category of Ca2+ sensors. Along with this, the protein can bind to a model target peptide (melittin) in a Ca2+-dependent manner

Disrupters of the Thymidylate Synthase Homodimer Accelerate Its Proteasomal Degradation and Inhibit Cancer Growth.

Drugs that target human thymidylate synthase (hTS) are widely used in anti-cancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces its over-expression and the development of drug resistance. We thus pursued an alternative strategy that led to the discovery of TS-dimer disrupters that bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. We performed a structural, spectroscopic and kinetic investigation of the effects of these small molecules andthe best one, E7, accelerates the proteasomal degradation of hTS in cancer cells. E7 showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers.</p