Generation of a laminopathies-specific iPSC line EHTJUi005-A-3 with homozygous knockout of the LMNA gene by CRISPR/Cas9 technology.

LAMIN A/C, encoded by the LMNA gene, supports the normal structure of the cell nucleus and regulates the connection between the nucleus and the cytoskeleton as a component of the nucleus envelope. The loss of expression and function of the LMNA gene would lead to the occurrence of congenital muscular dystrophy and Emery-Dreifuss muscular dystrophy which are collectively named as laminopathies. Here, we report a human induced pluripotent stem cell (iPSC) line (EHTJUi005-A-3) generated from a wild iPSC (EHTJUi005-A) with homozygous knockout of the gene LMNA through CRISPR/Cas9. This iPSC line provides a useful research model for studying laminopathies disease.

Brain-specific regulator of G-protein signaling 9-2 selectively interacts with alpha-actinin-2 to regulate calcium-dependent inactivation of NMDA receptors.

Regulator of G-protein signaling 9-1 (RGS9-1) and RGS9-2 are highly related RGS proteins with distinctive C termini arising from alternative splicing of RGS9 gene transcripts. RGS9-1 is expressed in photoreceptors where it functions as a regulator of transducin. In contrast, RGS9-2 is abundantly expressed in the brain, especially in basal ganglia, where its specific function remains poorly understood. To gain insight into the function of RGS9-2, we screened a human cDNA library for potential interacting proteins. This screen identified a strong interaction between RGS9-2 and alpha-actinin-2, suggesting a possible functional relationship between these proteins. Consistent with this idea, RGS9-2 and alpha-actinin-2 coimmunoprecipitated after coexpression in human embryonic kidney 293 (HEK-293) cells. Furthermore, endogenous RGS9-2 and alpha-actinin-2 could also be coimmunoprecipitated from extracts of rat striatum, an area highly enriched in both these proteins. These results supported the idea that RGS9-2 and alpha-actinin-2 could act in concert in central neurons. Like alpha-actinin-2, RGS9-2 coimmunoprecipitated NMDA receptors from striatal extracts, suggesting an interaction between RGS9-2, alpha-actinin-2, and NMDA receptors. Previous studies have shown that alpha-actinin mediates calcium-dependent inactivation of NMDA receptors. In HEK-293 cells expressing NMDA receptors, expression of RGS9-2 significantly modulated this form of NMDA receptor inactivation. Furthermore, this modulation showed remarkable preference for NMDA receptor inactivation mediated by alpha-actinin-2. Using a series of deletion constructs, we localized this effect to the RGS domain of the protein. These results identify an unexpected functional interaction between RGS9-2 and alpha-actinin-2 and suggest a potential novel role for RGS9-2 in the regulation of NMDA receptor function.

Electronic properties of multifurcated bent hydrogen bonds CH3...Y and CH2...Y.

H-bonding angle angleYHX has an important effect on the electronic properties of the H-bond Y...HX, such as intra- and intermolecular hyperconjugations and rehybridization, and topological properties of electron density. We studied the multifurcated bent H-bonds of the proton donors H3CZ (Z = F, Cl, Br), H2CO and H2CF2 with the proton acceptors Cl(-) and Br(-) at the four high levels of theory: MP2/6-311++G(d,p), MP2/6-311++G(2df,2p), MP2/6-311++G(3df,3pd) and QCISD/6-311++G(d,p), and found that they are all blue-shifted. These complexes have large interaction energies, 7-12 kcal mol(-1), and large blue shifts, delta r(HC) = -0.0025 --0.006 A and delta v(HC) = 30-90 cm(-1). The natural bond orbital analysis shows that the blue shifts of these H-bonds Y...HnCZ are mainly caused by three factors: rehybridization; indirect intermolecular hyperconjugation n(Y) -->sigma*(CZ), in that the electron density from n(Y) of the proton acceptor is transferred not to sigma*(CH), but to sigma*(CZ) of the donor; intramolecular hyperconjugation n(Z) -->sigma*(CH), in that the electron density in sigma*(CH) comes back to n(Z) of the donor such that the occupancy in sigma*(CH) decreases. The topological properties of the electron density of the bifurcated H-bonds Y...H2CZ are similar to those of the usual linear H-bonds, there is a bond critical point between Y and each hydrogen, and a ring critical point inside the tetragon YHCH. However, the topological properties of electron density of the trifurcated H-bonds Y...H3CZ are essentially different from those of linear H-bonds, in that the intermolecular bond critical point, which represents a closed-shell interaction, is not between Y and hydrogen, but between Y and carbon.