Significance: The transport of ions through the plant cell membrane establishes the key physicochemical parameters for cell function. Stress situations such as those created by soil salinity or low potassium conditions alter the ion transport across the membrane producing dramatic changes in the cell turgor, the membrane potential, and the intracellular pH and concentrations of toxic cations such as sodium and lithium. As a consequence, fundamental metabolic routes are inhibited. The CIPK family of 26 protein kinases regulates the function of several ion transporters at the cell membrane to restore ion homeostasis under stress situations. Our analyses provide an explanation on how the CIPKs are differentially activated to coordinate the adequate cell response to a particular stress.

Regulation of ion transport in plants is essential for cell function. Abiotic stress unbalances cell ion homeostasis, and plants tend to readjust it, regulating membrane transporters and channels. The plant hormone abscisic acid (ABA) and the second messenger Ca²⁺ are central in such processes, as they are involved in the regulation of protein kinases and phosphatases that control ion transport activity in response to environmental stimuli. The identification and characterization of the molecular mechanisms underlying the effect of ABA and Ca²⁺ signaling pathways on membrane function are central and could provide opportunities for crop improvement. The C2-domain ABA-related (CAR) family of small proteins is involved in the Ca²⁺-dependent recruitment of the pyrabactin resistance 1/PYR1-like (PYR/PYL) ABA receptors to the membrane. However, to fully understand CAR function, it is necessary to define a molecular mechanism that integrates Ca²⁺ sensing, membrane interaction, and the recognition of the PYR/PYL interacting partners. We present structural and biochemical data showing that CARs are peripheral membrane proteins that functionally cluster on the membrane and generate strong positive membrane curvature in a Ca²⁺-dependent manner. These features represent a mechanism for the generation, stabilization, and/or specific recognition of membrane discontinuities. Such structures may act as signaling platforms involved in the recruitment of PYR/PYL receptors and other signaling components involved in cell responses to stress.

The regulatory kinase CIPK23 borders on the ankyrin domain surface of the AKT1 channel in regulating cellular requirements for potassium.

Significance: Neurons coregulate their number of synapses and the probability of neurotransmitter release per synapse in an antagonistic manner. The binding of neuronal calcium sensor 1 (NCS-1) to the guanine exchange factor protein Ric8a coregulates these neuronal features. This study identified a small molecule, the phenothiazine FD44, that binds the interaction surface between NCS-1 and Ric8a, preventing the formation of the complex. Tested on a Drosophila model of the fragile X syndrome, where the number of synapses is in excess, FD44 proves effective to reduce synapse number to normal levels and restore normal learning performance. Our structure–function study shows the specificity of this compound and the drugability of the NCS-1/Ric8a interface for the treatment of fragile X and possibly, other synaptopathies.

Background: Biological systems respond to changes in both the Earth's magnetic and gravitational fields, but as experiments in space are expensive and infrequent, Earth-based simulation techniques are required. A high gradient magnetic field can be used to levitate biological material, thereby simulating microgravity and can also create environments with a reduced or an enhanced level of gravity (g), although special attention should be paid to the possible effects of the magnetic field (B) itself.Results: Using diamagnetic levitation, we exposedArabidopsis thaliana in vitrocallus cultures to five environments with different levels of effective gravity and magnetic field strengths. The environments included levitation, i.e. simulated μg* (close to 0g* atB= 10.1 T), intermediateg* (0.1g* atB= 14.7 T) and enhanced gravity levels (1.9g* atB= 14.7 T and 2g* atB= 10.1 T) plus an internal 1g* control (B= 16.5 T). The asterisk denotes the presence of the background magnetic field, as opposed to the effective gravity environments in the absence of an applied magnetic field, created using a Random Position Machine (simulated μg) and a Large Diameter Centrifuge (2g).Microarray analysis indicates that changes in the overall gene expression of cultured cells exposed to these unusual environments barely reach significance using an FDR algorithm. However, it was found that gravitational and magnetic fields produce synergistic variations in the steady state of the transcriptional profile of plants. Transcriptomic results confirm that high gradient magnetic fields (i.e. to create μg* and 2g* conditions) have a significant effect, mainly on structural, abiotic stress genes and secondary metabolism genes, but these subtle gravitational effects are only observable using clustering methodologies.Conclusions: A detailed microarray dataset analysis, based on clustering of similarly expressed genes (GEDI software), can detect underlying global-scale responses, which cannot be detected by means of individual gene expression techniques using raw or corrected p values (FDR). A subtle, but consistent, genome-scale response to hypogravity environments was found, which was opposite to the response in a hypergravity environment.

Protein stability is a fundamental issue in biomedical and biotechnological applications of proteins. Among these applications, gene- and enzyme-replacement strategies are promising approaches to treat inherited diseases that may benefit from protein engineering techniques, even though these beneficial effects have been largely unexplored. In the present study we apply a sequence-alignment statistics procedure (consensus-based approach) to improve the activity and stability of the human AGT (alanine–glyoxylate aminotransferase) protein, an enzyme which causes PH1 (primary hyperoxaluria type I) upon mutation. By combining only five consensus mutations, we obtain a variant (AGT-RHEAM) with largely enhanced in vitro thermal and kinetic stability, increased activity, and with no side effects on foldability and peroxisomal targeting in mammalian cells. The structure of AGT-RHEAM reveals changes at the dimer interface and improved electrostatic interactions responsible for increased kinetic stability. Consensus-based variants maintained the overall protein fold, crystallized more easily and improved the expression as soluble proteins in two different systems [AGT and CIPK24 (CBL-interacting serine/threonine-protein kinase) SOS2 (salt-overly-sensitive 2)]. Thus the consensus-based approach also emerges as a simple and generic strategy to increase the crystallization success for hard-to-get protein targets as well as to enhance protein stability and function for biomedical applications.