The Shank/ProSAP family of multidomain proteins is known to play an important role in organizing synaptic multiprotein complexes. Here we report a novel interaction between Shank and ␤PIX, a guanine nucleotide exchange factor for the Rac1 and Cdc42 small GTPases. This interaction is mediated by the PDZ domain of Shank and the C-terminal leucine zipper domain and the PDZ domain-binding motif at the extreme C terminus of ␤PIX. Shank colocalizes with ␤PIX at excitatory synaptic sites in cultured neurons. In brain, Shank forms a complex with ␤PIX and ␤PIX-associated signaling molecules including p21-associated kinase (PAK), an effector kinase of Rac1/Cdc42. Importantly, overexpression of Shank in cultured neurons promotes synaptic accumulation of ␤PIX and PAK. Considering the involvement of Rac1 and PAK in spine dynamics, these results suggest that Shank recruits ␤PIX and PAK to spines for the regulation of postsynaptic structure.Dendritic spines are actin-rich morphological specializations in neurons that mediate most excitatory synaptic transmission (1-3). The postsynaptic density (PSD) 1 is a microscopic structure within dendritic spines that is associated with the postsynaptic membrane and contains a variety of scaffolding and signaling proteins (4, 5).The Shank/ProSAP/SSTRIP family of multidomain proteins (Shank1, Shank2, and Shank3) plays important roles in organizing the PSD (6, 7). Shank is a relatively large protein (ϳ200 kDa) and contains various protein interaction domains including, from the N terminus, ankyrin repeats, an SH3 domain, a PDZ domain, a long (Ͼ1000 aa residues) proline-rich region and a SAM domain. The ankyrin repeats interact with ␣-fodrin, an actin-regulating protein, and Sharpin, a protein implicated in Shank multimerization (8, 9). The Shank PDZ domain interacts with the GKAP/SAPAP family of synaptic scaffold proteins and various membrane proteins including the calciumindependent receptor for latrotoxin, somatostatin receptors, and metabotropic glutamate receptors (10 -16). The long proline-rich region of Shank associates with IRSp53 (an insulin receptor tyrosine kinase substrate protein), Homer (an immediate early gene product that binds the group I metabotropic receptors and inositol 1,4,5-trisphosphate receptors), dynamin (a GTPase that regulates endocytosis), and cortactin (a regulator of the cortical actin cytoskeleton) (16 -20). The C-terminal SAM domain mediates multimerization of Shank proteins (10). There are several splice variants of Shank with alternative translational start and stop codons, suggesting that the Shank protein interactions are regulated by alternative splicing (11,12,21,22).Functionally, Shank is involved in the morphogenesis of dendritic spines (3, 23). Overexpression of Shank proteins promotes the maturation of spines in cultured neurons (24). The enhanced spine maturation by Shank requires the interaction of Shank with Homer, a protein that binds to metabotropic glutamate receptors and inositol 1,4,5-trisphosphate receptors (16). In addition, expression of ...

This study showed that tDC therapy in a preclinical model of MI was potentially translatable into an antiremodeling therapy for ischemic tissue repair.

In non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations, acquired resistance to EGFR-tyrosine kinase inhibitors (EGFR-TKI) can occur through a generation of bypass signals such as MET or AXL activation. In this study, we investigated the antitumor activity of NPS-1034, a newly developed drug that targets both MET and AXL, in NSCLC cells with acquired resistance to gefitinib or erlotinib (HCC827/GR and HCC827/ER, respectively). Characterization of H820 cells and evaluation of NPS-1034 efficacy in these cells were also performed. The resistance of HCC827/GR was mediated by MET activation, whereas AXL activation led to resistance in HCC827/ER. The combination of gefitinib or erlotinib with NPS-1034 synergistically inhibited cell proliferation and induced cell death in both resistant cell lines. Accordingly, suppression of Akt was noted only in the presence of treatment with both drugs. NPS-1034 was also effective in xenograft mouse models of HCC827/GR. Although the H820 cell line was reported previously to have T790M and MET amplification, we discovered that AXL was also activated in this cell line. There were no antitumor effects of siRNA or inhibitors specific for EGFR or MET, whereas combined treatment with AXL siRNA or NPS-1034 and EGFR-TKIs controlled H820 cells, suggesting that AXL is the main signal responsible for resistance. In addition, NPS-1034 inhibited cell proliferation as well as ROS1 activity in HCC78 cells with ROS1 rearrangement. Our results establish the efficacy of NPS-1034 in NSCLC cells rendered resistant to EGFR-TKIs because of MET or AXL activation or ROS1 rearrangement. Cancer Res; 74(1); 253-62. Ó2013 AACR.

Sindbis virus (SINV), the prototype alphavirus, contains a macro domain in the highly conserved N-terminal region of nonstructural protein 3 (nsP3). However, the biological role of the macro domain is unclear. Mutations of amino acids 10 and 24 from asparagine to alanine in the ADP-ribose binding region of the macro domain impaired SINV replication and viral RNA synthesis particularly in neurons, but did not alter binding of poly(ADP-ribose). Mutation at position 10 had the greatest effect and caused nsP3 instability in neurons, decreased SINV-induced death of mature, but not immature neurons, and attenuated virulence in 2 week-old, but not 5 day-old mice. A compensatory mutation at amino acid 31 in the macro domain of nsP3, as well as reversion of mutated amino acid 10, occurred during replication of double mutant SINV in vitro and in vivo. The nsP3 macro domain is important for SINV replication and age-dependent susceptibility to encephalomyelitis.

Stargazin is a transmembrane protein that interacts with AMPARs 1 and regulates their synaptic targeting (1, 2). The stargazer, a spontaneous mutant mouse (3) with defects in the stargazin gene (Cacng2) (4), displays an absence seizure (also known as petit-mal or spike-wave) and, as the name implies, a head-tossing movement, probably because of a defect in the vestibular system (3). In addition, stargazer mice develop an ataxic gait (3) and severe impairment in classical eye-blink conditioning (5), probably because of a cerebellar malfunction. Both mRNA and protein levels of brain-derived neurotrophic factor are selectively reduced in cerebellar granule cells of stargazer mice (5, 6). Stargazin, a protein with a calculated molecular mass of 36 kDa, contains four putative transmembrane domains and a cytosolic C terminus, and its primary structure is closely related to that of the ␥ subunits of voltagegated calcium channels (7-10). Stargazin (or ␥-2) associates with neuronal calcium channel subunits in vivo (11) and inhibits calcium channel activity by increasing steady-state inactivation (4,7,11,12).The functional association between stargazin and AMPAR was initially ascertained by the observation that postsynaptic AMPAR currents are selectively impaired in cerebellar granule cells of stargazer mice (13). A subsequent study revealed that stargazin mediates synaptic targeting of AMPARs by two distinct mechanisms (14). Stargazin initially interacts with AMPARs and assists their translocation to the extrasynaptic surface membrane. Next, the AMPAR-stargazin complex is targeted to synaptic sites by binding to PSD-95 and related PDZ proteins. In support of this hypothesis, a stargazin mutant lacking the last four residues (stargazin⌬C) rescues extrasynaptic but not synaptic AMPAR currents in cerebellar granule cells of stargazer mice (14). However, little is known about whether the stargazin-mediated synaptic targeting of AMPARs is regulated and, if so, what these regulatory mechanisms involve.The C terminus of stargazin contains the end sequence RRT-TPV, which belongs to the class I PDZ-binding motif, (S/T)XV (S/T, Ser or Thr; X, any aa residue; V, hydrophobic residue) (15-17). Interestingly, the RRTT sequence of the C terminus additionally corresponds to the consensus sequence for phosphorylation by PKA, (R/K)(R/X)X(S/T), suggesting that Thr at the Ϫ2 position (RRTTPV, designated T321) is phosphorylated by PKA. The crystal structure of the PDZ3 domain of PSD-95 (class I) complexed with the C terminus of CRIPT, a PSD-95-binding protein that ends with the QTSV sequence (18), reveals that the Thr residue at the Ϫ2 position interacts with His-372 of PDZ3 (15). Specifically, the hydroxyl oxygen of the Thr forms a hydrogen bond with the N-3 nitrogen of His-372. Therefore, phosphorylation of T321 at the stargazin C terminus may weaken the interaction between stargazin and the PDZ domains of PSD-95. Consistently, earlier data demonstrate that phosphorylation of the Ser residue at the Ϫ2 position of Kir2.3 (an inward rectifie...

In Bacillus subtilis, large genomic deletions have been carried out for genome reduction, antibiotic overproduction, and heterologous protein overexpression. In view of the ecofriendliness of B. subtilis, it is critical that engineering preserves its food-grade status and avoids leaving foreign DNA in the genome. Existing methods of generating large genomic deletions leave antibiotic resistance markers or display low mutation efficiency. In this study, we introduced a clustered regularly interspaced short palindromic repeat-derived genome engineering technique to develop a highly efficient method of generating large genomic deletions in B. subtilis without any trace of foreign DNA. Using our system, we produced 38 kb plipastatin-synthesizing pps operon deletion with 80% efficiency. The significant increase in mutation efficiency was due to plasmids-delivered Streptococcus pyogenes-originated SpCas9, target-specific sgRNA and a donor DNA template, which produces SpCas9/sgRNA endonuclease complex continuously for attacking target chromosome until the mutagenic repair occurs. Our system produced single-gene deletion in spo0A (∼100%), point mutation (∼68%) and GFP gene insertion (∼97%) in sigE and demonstrated its broad applicability for various types of site-directed mutagenesis in B. subtilis.

Heart failure (HF) is a frequent consequence of myocardial infarction (MI). Identification of the precise, time-dependent composition of inflammatory cells may provide clues for the establishment of new biomarkers and therapeutic approaches targeting post-MI HF. Here, we investigate the spatiotemporal dynamics of MI-associated immune cells in a mouse model of MI using spatial transcriptomics and single-cell RNA-sequencing (scRNA-seq). We identify twelve major immune cell populations; their proportions dynamically change after MI. Macrophages are the most abundant population at all-time points (>60%), except for day 1 post-MI. Trajectory inference analysis shows upregulation of Trem2 expression in macrophages during the late phase post-MI. In vivo injection of soluble Trem2 leads to significant functional and structural improvements in infarcted hearts. Our data contribute to a better understanding of MI-driven immune responses and further investigation to determine the regulatory factors of the Trem2 signaling pathway will aid the development of novel therapeutic strategies for post-MI HF.

Coordination of neocortical oscillations has been hypothesized to underlie the “binding” essential to cognitive function. However, the mechanisms that generate neocortical oscillations in physiological frequency bands remain unknown. We hypothesized that interlaminar relations in neocortex would provide multiple intermediate loops that would play particular roles in generating oscillations, adding different dynamics to the network. We simulated networks from sensory neocortex using nine columns of event-driven rule-based neurons wired according to anatomical data and driven with random white-noise synaptic inputs. We tuned the network to achieve realistic cell firing rates and to avoid population spikes. A physiological frequency spectrum appeared as an emergent property, displaying dominant frequencies that were not present in the inputs or in the intrinsic or activated frequencies of any of the cell groups. We monitored spectral changes while using minimal dynamical perturbation as a methodology through gradual introduction of hubs into individual layers. We found that hubs in layer 2/3 excitatory cells had the greatest influence on overall network activity, suggesting that this subpopulation was a primary generator of theta/beta strength in the network. Similarly, layer 2/3 interneurons appeared largely responsible for gamma activation through preferential attenuation of the rest of the spectrum. The network showed evidence of frequency homeostasis: increased activation of supragranular layers increased firing rates in the network without altering the spectral profile, and alteration in synaptic delays did not significantly shift spectral peaks. Direct comparison of the power spectra with experimentally recorded local field potentials from prefrontal cortex of awake rat showed substantial similarities, including comparable patterns of cross-frequency coupling.