Working out cabling and also plasticity in synaptic groupings

These effects were associated with reduced lipolytic activity because of downregulation of lipolytic enzymes such as adipose triglyceride lipase and hormone-sensitive lipase in adipose tissue of the conditional knockout mice. Given that, among GPR6CA ligands tested, GluOC and ornithine increased the expression of adipose triglyceride lipase in cultured 3T3-L1 adipocytes in a manner dependent on GPRC6A, our results suggest that the constitutive activation of GPRC6A signaling in adipocytes by GluOC or ornithine plays a key role in adipose lipid handling and the prevention of obesity and related metabolic disorders.Proline and arginine-rich end leucine-rich repeat protein (PRELP) is a member of the small leucine-rich repeat proteoglycans (SLRPs) family. Levels of PRELP mRNA are downregulated in many types of cancer, and PRELP has been reported to have suppressive effects on tumor cell growth, although the molecular mechanism has yet to be fully elucidated. Given that other SLRPs regulate signaling pathways through interactions with various membrane proteins, we reasoned that PRELP likely interacts with membrane proteins to maintain cellular homeostasis. To identify membrane proteins that interact with PRELP, we carried out coimmunoprecipitation coupled with mass spectrometry (CoIP-MS). We prepared membrane fractions from Expi293 cells transfected to overexpress FLAG-tagged PRELP or control cells and analyzed samples precipitated with anti-FLAG antibody by mass spectrometry. Comparison of membrane proteins in each sample identified several that seem to interact with PRELP; among them, we noted two growth factor receptors, insulin-like growth factor I receptor (IGFI-R) and low-affinity nerve growth factor receptor (p75NTR), interactions with which might help to explain PRELP's links to cancer. We demonstrated that PRELP directly binds to extracellular domains of these two growth factor receptors with low micromolar affinities by surface plasmon resonance analysis using recombinant proteins. Furthermore, cell-based analysis using recombinant PRELP protein showed that PRELP suppressed cell growth and affected cell morphology of A549 lung carcinoma cells, also at micromolar concentration. Selleckchem NVP-DKY709 These results suggest that PRELP regulates cellular functions through interactions with IGFI-R and p75NTR and provide a broader set of candidate partners for further exploration.Aggregation of α-synuclein (αS) leads to the hallmark neuropathology of Parkinson's disease (PD) and related synucleinopathies. αS has been described to exist in both cytosolic and membrane-associated forms, the relative abundance of which has remained unsettled. To study αS under the most relevant conditions by a quantitative method, we cultured and matured rodent primary cortical neurons for >17 days and determined αS cytosolmembrane distribution via centrifugation-free sequential extractions based on the weak ionic detergent digitonin. We noticed that at lower temperatures (4 °C or room temperature), αS was largely membrane-associated. At 37 °C, however, αS solubility was markedly increased. In contrast, the extraction of control proteins (GAPDH, cytosolic; calnexin, membrane) was not affected by temperature. When we compared the relative distribution of the synuclein homologs αS and β-synuclein (βS) under various conditions that differed in temperature and digitonin concentration (200-1200 μg/ml), we consistently found αS to be more membrane-associated than βS. Both proteins, however, exhibited temperature-dependent membrane binding. Under the most relevant conditions (37 °C and 800 μg/ml digitonin, i.e., the lowest digitonin concentration that extracted cytosolic GAPDH to near completion), cytosolic distribution was 49.8% ± 9.0% for αS and 63.6% ± 6.6% for βS. PD-linked αS A30P was found to be largely cytosolic, confirming previous studies that had used different methods. Our work highlights the dynamic nature of cellular synuclein behavior and has important implications for protein-biochemical and cell-biological studies of αS proteostasis, such as testing the effects of genetic and pharmacological manipulations.The mitogen-responsive, ETS-domain transcription factor ELK-1 stimulates the expression of immediate early genes at the onset of the cell cycle and participates in early developmental programming. ELK-1 is subject to multiple levels of posttranslational control, including phosphorylation, SUMOylation, and ubiquitination. Recently, removal of monoubiquitin from the ELK-1 ETS domain by the Ubiquitin Specific Protease USP17 was shown to augment ELK-1 transcriptional activity and promote cell proliferation. Here we have used coimmunoprecipitation experiments, protein turnover and ubiquitination assays, RNA-interference and gene expression analyses to examine the possibility that USP17 acts antagonistically with the F-box protein FBXO25, an E3 ubiquitin ligase previously shown to promote ELK-1 ubiquitination and degradation. Our data confirm that FBXO25 and ELK-1 interact in HEK293T cells and that FBXO25 is active toward Hand1 and HAX1, two of its other candidate substrates. However, our data indicate that FBXO25 neither promotes ubiquitination of ELK-1 nor impacts on its transcriptional activity and suggest that an E3 ubiquitin ligase other than FBXO25 regulates ELK-1 ubiquitination and function.Pseudomonas aeruginosa is an opportunistic pathogen requiring iron for its survival and virulence. P. aeruginosa can acquire iron from heme via the nonredundant heme assimilation system and Pseudomonas heme uptake (Phu) systems. Heme transported by either the heme assimilation system or Phu system is sequestered by the cytoplasmic protein PhuS. Furthermore, PhuS has been shown to specifically transfer heme to the iron-regulated heme oxygenase HemO. As the PhuS homolog ShuS from Shigella dysenteriae was observed to bind DNA as a function of its heme status, we sought to further determine if PhuS, in addition to its role in regulating heme flux through HemO, functions as a DNA-binding protein. Herein, through a combination of chromatin immunoprecipitation-PCR, EMSA, and fluorescence anisotropy, we show that apo-PhuS but not holo-PhuS binds upstream of the tandem iron-responsive sRNAs prrF1,F2. Previous studies have shown the PrrF sRNAs are required for sparing iron for essential proteins during iron starvation.