Conditional switching of KIF2A mutation provides new insights into cortical malformation pathogeny.

By using the Cre-mediated genetic switch technology, we were able to successfully generate a conditional knock-in mouse, bearing the KIF2A p.His321Asp missense point variant, identified in a subject with malformations of cortical development. These mice present with neuroanatomical anomalies and microcephaly associated with behavioral deficiencies and susceptibility to epilepsy, correlating with the described human phenotype. Using the flexibility of this model, we investigated RosaCre-, NestinCre- and NexCre-driven expression of the mutation to dissect the pathophysiological mechanisms underlying neurodevelopmental cortical abnormalities. We show that the expression of the p.His321Asp pathogenic variant increases apoptosis and causes abnormal multipolar to bipolar transition in newborn neurons, providing therefore insights to better understand cortical organization and brain growth defects that characterize KIF2A-related human disorders. We further demonstrate that the observed cellular phenotypes are likely to be linked to deficiency in the microtubule depolymerizing function of KIF2A.

Truncated Actin-Targeting Macrolide Derivative Blocks Cancer Cell Motility and Invasion of Extracellular Matrix.

Cancer metastasis is a complex process involving highly motile tumor cells that breach tissue barriers, enter the bloodstream and lymphatic system, and disseminate throughout the body as circulating tumor cells. The primary cellular mechanism contributing to these critical events is the reorganization of the actin cytoskeleton. Mycalolide B (MycB) is an actin-targeting marine macrolide that can suppress proliferation, migration, and invasion of breast and ovarian cancer cells at low nanomolar doses. Through structure-activity relationship studies focused on the actin-binding tail region (C24-C35) of MycB, we identified a potent truncated derivative that inhibits polymerization of G-actin and severs F-actin by binding to actin's barbed end cleft. Biological analyses of this miniature MycB derivative demonstrate that it causes a rapid collapse of the actin cytoskeleton in ovarian cancer cells and impairs cancer cell motility and invasion of the extracellular matrix (ECM) by inhibiting invadopodia-mediated ECM degradation. These studies provide essential proof-of-principle for developing actin-targeting therapeutic agents to block cancer metastasis and establish a synthetically tractable barbed end-binding pharmacophore that can be further improved by adding targeting groups for precision drug design.

Structural Studies of Multidrug Resistance Protein 1 Using "Almost" Cysless Template.

Multidrug resistance protein, MRP1 (ABCC1) is a broad-spectrum ATP-binding cassette transporter that plays a major role in defense against dietary and environmental toxicants, in addition to contributing toward multidrug resistance of certain types of malignancy. Elucidating the molecular structure of hMRP1 is key to determining its mechanism of substrate recognition and transport. Here, we report the first successful attempt using cysteine-scanning mutagenesis coupled with cross-linking studies to probe the structure of hMRP1 in its native environment of the cell membrane or in membrane vesicles. We have established that an active 3Cys ΔMRP1 (MRP204-1531) mutant, described in previous studies from our laboratory, is a suitable template with which to generate single- and double-cysteine mutants for performing cysteine mutagenesis studies. We have now used 3Cys ΔMRP1 to probe the arrangement of several TM segments, as well as the location of individual amino acids in these regions. Cysteine residues were introduced into TMs 8, 14, 15, and 16 of 3Cys ΔMRP1. The mutants were then subjected to chemical cross-linking analyses, and cross-linking was detected between the following cysteine pairs: Cys388 (TM7) and I1193C (TM16); Cys388 (TM7) and E1144C (TM15); R433C (TM8) and E1144C (TM15); and R433C (TM8) and T1082C (TM14). The aqueous accessibility of these residues and the possible implications of the differences between the open and closed states of the protein are also discussed. Moreover, using competition experiments involving a well characterized substrate and a cross-linking reagent for probing the Cys388/ I1193C mutant, we have defined these amino acid positions as a component of the potential site for estrone sulfate binding.

Toward a Template for Synthetic Actin-Targeting Macrolide Analogues That Inhibit Cancer Cell Invasiveness.

Actin barbed end-binding macrolides have been shown to inhibit cancer cell motility and invasion of extracellular matrix (ECM), evoking their potential utility as therapies for metastatic cancers. Unfortunately, the direct use of these compounds in clinical settings is impeded by their limited natural abundance, challenging total synthesis, and detrimental effects on normal tissues. To develop potent analogues of these compounds that are simpler to synthesize and compatible with cell-specific targeting systems, such as antibodies, we designed over 20 analogues of the acyclic side chain (tail) of the macrolide Mycalolide B. These analogues probed the contributions of four distinct regions of the tail towards the inhibition of actin polymerization and ECM invasion by human lung cancer A549 cells. We observed that two of these regions tolerate considerable substituent variability, and we identified a specific combination of substituents that leads to the optimal inhibition of the ECM invasion activity of A549 cells.

Kinesin-8-specific loop-2 controls the dual activities of the motor domain according to tubulin protofilament shape.

Kinesin-8s are dual-activity motor proteins that can move processively on microtubules and depolymerize microtubule plus-ends, but their mechanism of combining these distinct activities remains unclear. We addressed this by obtaining cryo-EM structures (2.6-3.9 Å) of Candida albicans Kip3 in different catalytic states on the microtubule lattice and on a curved microtubule end mimic. We also determined a crystal structure of microtubule-unbound CaKip3-ADP (2.0 Å) and analyzed the biochemical activity of CaKip3 and kinesin-1 mutants. These data reveal that the microtubule depolymerization activity of kinesin-8 originates from conformational changes of its motor core that are amplified by dynamic contacts between its extended loop-2 and tubulin. On curved microtubule ends, loop-1 inserts into preceding motor domains, forming head-to-tail arrays of kinesin-8s that complement loop-2 contacts with curved tubulin and assist depolymerization. On straight tubulin protofilaments in the microtubule lattice, loop-2-tubulin contacts inhibit conformational changes in the motor core, but in the ADP-Pi state these contacts are relaxed, allowing neck-linker docking for motility. We propose that these tubulin shape-induced alternations between pro-microtubule-depolymerization and pro-motility kinesin states, regulated by loop-2, are the key to the dual activity of kinesin-8 motors.

Ste2 receptor-mediated chemotropism of Fusarium graminearum contributes to its pathogenicity against wheat.

Fusarium Head Blight of wheat, caused by the filamentous fungus Fusarium graminearum, leads to devastating global food shortages and economic losses. While many studies have addressed the responses of both wheat and F. graminearum during their interaction, the possibility of fungal chemotropic sensing enabling pathogenicity remains unexplored. Based on recent findings linking the pheromone-sensing G-protein-coupled receptor Ste2 to host-directed chemotropism in Fusarium oxysporum, we investigated the role of the Ste2 receptor and its downstream signaling pathways in mediating chemotropism of F. graminearum. Interestingly, a chemotropic response of growing hyphae towards catalytically active Triticum aestivum 'Roblin' cultivar secreted peroxidases was detected, with deletion of STE2 in F. graminearum leading to loss of the observed response. At the same time, deletion of STE2 significantly decreased infection on germinating wheat coleoptiles, highlighting an association between Ste2, chemotropism and infection by F. graminearum. Further characterization revealed that the peroxidase-directed chemotropism is associated with stimulation of the fungal cell wall integrity mitogen-activated protein kinase signaling cascade. Altogether, this study demonstrates conservation of Ste2-mediated chemotropism by Fusarium species, and its important role in mediating pathogenicity.

Ternary complex of Kif2A-bound tandem tubulin heterodimers represents a kinesin-13-mediated microtubule depolymerization reaction intermediate.

Kinesin-13 proteins are major microtubule (MT) regulatory factors that catalyze removal of tubulin subunits from MT ends. The class-specific "neck" and loop 2 regions of these motors are required for MT depolymerization, but their contributing roles are still unresolved because their interactions with MT ends have not been observed directly. Here we report the crystal structure of a catalytically active kinesin-13 monomer (Kif2A) in complex with two bent αß-tubulin heterodimers in a head-to-tail array, providing a view of these interactions. The neck of Kif2A binds to one tubulin dimer and the motor core to the other, guiding insertion of the KVD motif of loop 2 in between them. AMPPNP-bound Kif2A can form stable complexes with tubulin in solution and trigger MT depolymerization. We also demonstrate the importance of the neck in modulating ATP turnover and catalytic depolymerization of MTs. These results provide mechanistic insights into the catalytic cycles of kinesin-13.

Spraying enzymes in microemulsions of AOT in nonpolar organic solvents for fabrication of enzyme electrodes.

A new technique suitable for automated, large-scale fabrication of enzyme electrodes by air-spraying enzymes in organic inks is presented. Model oxidoreductases, tyrosinase (Tyr) and glucose oxidase (GOx), were adapted to octane-based ink by entrapment in a system of reverse micelles (RM) of surfactant AOT in octane to separate and stabilize the catalytically active forms of the enzymes in nonpolar organic media. Nonpolar caoutchouk polymer was also used to create a kind of "dry micelles" at the electrode/solution interface. Enzyme/RM/polymer-containing organic inks were air-brushed onto conductive supports and were subsequently covered by sprayed Nafion membranes. The air-brushed enzyme electrodes exhibited relevant bioelectrocatalytic activity toward catechol and glucose, with a linear detection range of 0.1-100 microM catechol and 0.5-7 mM glucose; the sensitivities were 2.41 A M(-1) cm(-2) and 2.98 mA M(-1) cm(-2) for Tyr and GOx electrodes, respectively. The proposed technique of air-brushing enzymes in organic inks enables automated construction of disposable enzyme electrodes of various designs on a mass-production scale.

Modification of beta-lactoglobulin by oligofructose: impact on protein adsorption at the air-water interface.

Maillard products of beta-lactoglobulin (betaLg) and fructose oligosaccharide (FOS) were obtained in different degrees of modification depending on incubation time and pH. By use of a variety of biochemical and spectroscopic tools, it was demonstrated that the modification at limited degrees does not significantly affect the secondary, tertiary, and quaternary structure of betaLg. The consequence of the modification on the thermodynamics of the protein was studied using differential scanning calorimetry, circular dichroism, and by monitoring the fluorescence intensity of protein samples with different concentrations of guanidine-HCl. The modification leads to lowering of the denaturation temperature by 5 degrees C and a reduction of the free energy of stabilization of about 30%. Ellipsometry and drop tensiometry demonstrated that upon adsorption to air-water interfaces in equilibrium modified betaLg exerts a lower surface pressure than native betaLg (16 versus 22 mN/m). Moreover, the surface elastic modulus increased with increasing surface pressure but reached significantly smaller values in the case of FOS-betaLg. Compared to native betaLg, modification of the protein with oligofructose moieties results in higher surface loads and thicker surface layers. The consequences of these altered surface rheological properties are discussed in view of the functional behavior in technological applications.

Protein adsorption at air-water interfaces: a combination of details.

Using a variety of spectroscopic techniques, a number of molecular functionalities have been studied in relation to the adsorption process of proteins to air-water interfaces. While ellipsometry and drop tensiometry are used to derive information on adsorbed amount and exerted surface pressure, external reflection circular dichroism, infrared, and fluorescence spectroscopy provide, next to insight in layer thickness and surface layer concentration, molecular details like structural (un)folding, local mobility, and degree of protonation of carboxylates. It is shown that the exposed hydrophobicity of the protein or chemical reactivity of solvent-exposed groups may accelerate adsorption, while increased electrostatic repulsion slows down the process. Also aggregate formation enhances the fast development of a surface pressure. A more bulky appearance of proteins lowers the collision intensity in the surface layer, and thereby the surface pressure, while it is shown to be difficult to affect protein interactions within the surface layer on basis of electrostatic interactions. This work illustrates that the adsorption properties of a protein are a combination of molecular details, rather than determined by a single one.