SUMMARYMitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Modeling of these defects has been difficult because of the challenges associated with engineering mtDNA. We show here that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) retain the parental mtDNA profile and exhibit a metabolic switch toward oxidative phosphorylation. NPCs derived in this way from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) showed defective ATP production and abnormally high mitochondrial membrane potential (MMP), plus altered calcium homeostasis, which represents a potential cause of neural impairment. High-content screening of FDA-approved drugs using the MMP phenotype highlighted avanafil, which we found was able to partially rescue the calcium defect in patient NPCs and differentiated neurons. Overall, our results show that iPSC-derived NPCs provide an effective model for drug screening to target mtDNA disorders that affect the nervous system.

Biologically inert, photoactivatable precursors ("caged" compounds) of cyclic nucleoside monophosphates (cNMPs) are powerful tools for studying the spatiotemporal dynamics of cyclic nucleotide dependent processes. Among these compounds, (coumarin-4-yl)methyl esters of cNMPs are most useful because they show no background bioactivity, are stable to solvolysis, and can be photolyzed efficiently and extremely quickly. [1,2] Recently, we introduced [7-(diethylamino)coumarin-4-yl]methyl (DEACM) esters of cNMPs as caged compounds. [3,4] Compared with other coumarinylmethyl-caged cNMPs, the DEACM esters photorelease cNMPs with higher photosensitivity at long-wavelength irradiation (up to 436 nm), thus minimizing or even preventing damage to cellular components and chromophores by photobleaching.Herein, we describe the development of the 7-[bis(carboxymethyl)amino]-substituted coumarinylmethyl building blocks 1 and 2 (structures in Scheme 1) for the caging of phosphates and other functionalities. With the axial and the equatorial diastereomers of the {7-[bis(carboxymethyl)-amino]coumarin-4-yl}methyl (BCMACM) esters of cAMP, cGMP, 8-Br-cAMP, and 8-Br-cGMP 3-6 (Scheme 1), we present new variants of the DEACM-caged cNMPs, which maintain their favorable properties and additionally have much higher aqueous solubility by virtue of their anionic[*] Dr.

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GTPases of immunity-associated proteins (GIMAPs) are a distinctive family of GTPases, which control apoptosis in lymphocytes and play a central role in lymphocyte maturation and lymphocyte-associated diseases. To explore their function and mechanism, we determined crystal structures of a representative member, GIMAP2, in different nucleotide-loading and oligomerization states. Nucleotide-free and GDP-bound GIMAP2 were monomeric and revealed a guanine nucleotide-binding domain of the TRAFAC (translation factor associated) class with a unique amphipathic helix α7 packing against switch II. In the absence of α7 and the presence of GTP, GIMAP2 oligomerized via two distinct interfaces in the crystal. GTP-induced stabilization of switch I mediates dimerization across the nucleotide-binding site, which also involves the GIMAP specificity motif and the nucleotide base. Structural rearrangements in switch II appear to induce the release of α7 allowing oligomerization to proceed via a second interface. The unique architecture of the linear oligomer was confirmed by mutagenesis. Furthermore, we showed a function for the GIMAP2 oligomer at the surface of lipid droplets. Although earlier studies indicated that GIMAPs are related to the septins, the current structure also revealed a strikingly similar nucleotide coordination and dimerization mode as in the dynamin GTPase. Based on this, we reexamined the relationships of the septin-and dynamin-like GTPases and demonstrate that these are likely to have emerged from a common membrane-associated dimerizing ancestor. This ancestral property appears to be critical for the role of GIMAPs as nucleotide-regulated scaffolds on intracellular membranes. G protein | protein structure

A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3␤ interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3␤ (glycogen synthase kinase 3␤). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3␤ by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3␤ and thereby provides a mechanism for the integration of PKA and GSK3␤ signaling pathways. A-kinase anchoring proteins (AKAPs)3 are a family of scaffoldingproteinscharacterizedbytheabilitytobindcAMPdependent protein kinase (protein kinase A (PKA)). They tether PKA in the vicinity of its substrates, thereby facilitating their phosphorylation. In addition, AKAPs bind further signaling molecules, including other protein kinases (e.g. protein kinase C and protein kinase D), phosphodiesterases (e.g. PDE4D), and protein phosphatases (e.g. PP1 and PP2B/calcineurin). A few AKAPs possess catalytic activity. For example, AKAP-Lbc is a Rho guanine nucleotide exchange factor (1-3). Thus, AKAPs assemble multiprotein complexes and thereby coordinate cellular signaling. AKAPs are required for many cellular processes, including vasopressin-mediated water reabsorption in renal principal cells and ␤-adrenoreceptor-dependent increases of cardiac myocyte contractility (2, 4, 5).The PKA holoenzyme consists of a dimer of regulatory RI or RII subunits and two catalytic subunits, each bound to one R subunit. Upon binding of two molecules of cAMP to each R subunit, the catalytic subunits dissociate and phosphorylate their substrates (6, 7). The interaction of AKAPs with PKA is mediated by the PKA-anchoring domain of AKAPs and the dimerization and docking (DD) domain of R subunit dimers. Because most AKAPs preferentially anchor RII subunits, PKAanchoring domains are termed RII-binding domains (RIIBD). These domains are structurally conserved amphipathic helices, 14 -18 amino acid residues in length (8, 9). Based on recently described determinants of the RIIBD/DD domain interaction (8 -10), we developed a bioinformatics and peptide array screening approach to identify new AKAPs. For one of the discovered proteins, GSKIP (GSK3␤ interaction protein), we show that it functions as an AKAP.Mammalian cells express two isoforms of glycogen synthase kinase-3 (GSK3), GSK...