Insulin Resistance in Striated Muscle-specific Integrin Receptor ��1-deficient Mice

Integrin receptor plays key roles in mediating both inside-out and outside-in signaling between cells and the extracellular matrix. We have observed that the tissue-specific loss of the integrin β1 subunit in striated muscle results in a near complete loss of integrin β1 subunit protein expression concomitant with a loss of talin and to a lesser extent, a reduction in F-actin content. Muscle-specific integrin β1-deficient mice had no significant difference in food intake, weight gain, fasting glucose, and insulin levels with their littermate controls. However, dynamic analysis of glucose homeostasis using euglycemichyperinsulinemic clamps demonstrated a 44 and 48% reduction of insulin-stimulated glucose infusion rate and glucose clearance, respectively. The whole body insulin resistance resulted from a specific inhibition of skeletal muscle glucose uptake and glycogen synthesis without any significant effect on the insulin suppression of hepatic glucose output or insulin-stimulated glucose uptake in adipose tissue. The reduction in skeletal muscle insulin responsiveness occurred without any change in GLUT4 protein expression levels but was associated with an impairment of the insulin-stimulated protein kinase B/Akt serine 473 phosphorylation but not threonine 308. The inhibition of insulin-stimulated serine 473 phosphorylation occurred concomitantly with a decrease in integrin-linked kinase expression but with no change in the mTOR·Rictor·LST8 complex (mTORC2). These data demonstrate an in vivo crucial role of integrin β1 signaling events in mediating cross-talk to that of insulin action.Integrin receptors are a large family of integral membrane proteins composed of a single α and β subunit assembled into a heterodimeric complex. There are 19 α and 8 β mammalian subunit isoforms that combine to form 25 distinct α,β heterodimeric receptors (1-5). These receptors play multiple critical roles in conveying extracellular signals to intracellular responses (outside-in signaling) as well as altering extracellular matrix interactions based upon intracellular changes (inside-out signaling). Despite the large overall number of integrin receptor complexes, skeletal muscle integrin receptors are limited to seven α subunit subtypes (α1, α3, α4, α5, α6, α7, and αν subunits), all associated with the β1 integrin subunit (6, 7).Several studies have suggested an important cross-talk between extracellular matrix and insulin signaling. For example, engagement of β1 subunit containing integrin receptors was observed to increase insulin-stimulated insulin receptor substrate (IRS)² phosphorylation, IRS-associated phosphatidylinositol 3-kinase, and activation of protein kinase B/Akt (8-11). Integrin receptor regulation of focal adhesion kinase was reported to modulate insulin stimulation of glycogen synthesis, glucose transport, and cytoskeleton organization in cultured hepatocytes and myoblasts (12, 13). Similarly, the integrin-linked kinase (ILK) was suggested to function as one of several potential upstream kinases that phosphorylate and activate Akt (14-18). In this regard small interfering RNA gene silencing of ILK in fibroblasts and conditional ILK gene knockouts in macrophages resulted in a near complete inhibition of insulin-stimulated Akt serine 473 (Ser-473) phosphorylation concomitant with an inhibition of Akt activity and phosphorylation of Akt downstream targets (19). However, a complex composed of mTOR·Rictor·LST8 (termed mTORC2) has been identified in several other studies as the Akt Ser-473 kinase (20, 21). In addition to Ser-473, Akt protein kinase activation also requires phosphorylation on threonine 308 Thr-30 by phosphoinositide-dependent protein kinase, PDK1 (22-24).In vivo, skeletal muscle is the primary tissue responsible for postprandial (insulin-stimulated) glucose disposal that results from the activation of signaling pathways leading to the translocation of the insulin-responsive glucose transporter, GLUT4, from intracellular sites to the cell surface membranes (25, 26). Dysregulation of any step of this process in skeletal muscle results in a state of insulin resistance, thereby predisposing an individual for the development of diabetes (27-33). Although studies described above have utilized a variety of tissue culture cell systems to address the potential involvement of integrin receptor signaling in insulin action, to date there has not been any investigation of integrin function on insulin action or glucose homeostasis in vivo. To address this issue, we have taken advantage of Cre-LoxP technology to inactivate the β1 integrin receptor subunit gene in striated muscle. We have observed that muscle creatine kinase-specific integrin β1 knock-out (MCKItgβ1 KO) mice display a reduction of insulin-stimulated glucose infusion rate and glucose clearance. The impairment of insulin-stimulated skeletal muscle glucose uptake and glycogen synthesis resulted from a decrease in Akt Ser-473 phosphorylation concomitant with a marked reduction in ILK expression. Together, these data demonstrate an important cross-talk between integrin receptor function and insulin action and suggests that ILK may function as an Akt Ser-473 kinase in skeletal muscle.     

Identification of Residues in the Drug Translocation Pathway of the Human Multidrug Resistance P-glycoprotein by Arginine Mutagenesis

P-glycoprotein (P-gp, ATP-binding cassette B1) is a drug pump that extracts toxic drug substrates from the plasma membrane and catalyzes their ATP-dependent efflux. To map the residues in the drug translocation pathway, we performed arginine-scanning mutagenesis on all transmembrane (TM) segments (total = 237 residues) of a P-gp processing mutant (G251V) defective in folding (15% maturation efficiency) (glycosylation state used to monitor folding). The rationale was that arginines introduced into the drug-binding sites would mimic drug rescue and enhance maturation of wild-type or processing mutants of P-gp. It was found that 38 of the 89 mutants that matured had enhanced maturation. Enhancer mutations were found in 11 of the 12 TM segments with the largest number found in TMs 6 and 12 (seven in each), TMs that are critical for P-gp-drug substrate interactions. Modeling of the TM segments showed that the enhancer arginines were found on the hydrophilic face, whereas inhibitory arginines were located on a hydrophobic face that may be in contact with the lipid bilayer. It was found that many of the enhancer arginines caused large alterations in P-gp-drug interactions in ATPase assays. For example, mutants A302R (TM5), L339R (TM6), G872R (TM10), F942R (TM11), Q946R (TM11), V982R (TM12), and S993R (TM12) reduced the apparent affinity for verapamil by ∼10-fold, whereas the F336R (TM6) and M986R (TM12) mutations caused at least a 10-fold increase in apparent affinity for rhodamine B. The results suggest that P-gp contains a large aqueous-filled drug translocation pathway with multiple drug-binding sites that can accommodate the bulky arginine side chains to promote folding of the protein.The human multidrug resistance P-glycoprotein (P-gp, ATP-binding cassette B1)² is an ATP-dependent drug pump that mediates efflux of a broad range of hydrophobic compounds out of the cell (1). It is expressed in the epithelium of liver, kidney, and gastrointestinal tract and at the blood-brain or blood-testes barrier where it functions to protect us from cytotoxic compounds. It is clinically important because it contributes to multidrug resistance in diseases such as cancer and AIDS (1).P-gp is an ATP-binding cassette transporter of 1280 amino acids that consists of two homologous halves (2). Each half begins with a transmembrane domain (TMD) containing six TM segments followed by a nucleotide-binding domain (NBD).A key goal to understanding the mechanism of P-gp drug transport is to identify the amino acids that line the drug translocation pathway. Because P-gp extracts drug substrates from the lipid bilayer, the drug-binding pocket/drug translocation pathway are predicted to reside in the transmembrane (TM) segments. We previously showed that the TMDs alone were sufficient for drug binding (3). Expression of the TMDs as separate polypeptides showed that both TMD1 and TMD2 were required for binding drug substrate (4). The results of studies utilizing cysteine-scanning mutagenesis and labeling with thiol-reactive drug substrates suggested that all of the TM segments contribute to the drug-binding pocket/drug translocation pathway (reviewed in Ref. 5). The next step is to identify the specific amino acids that line the drug translocation pathway. It is important to identify amino acids that line the drug translocation pathway and to compare whether the biochemical evidence supports a model of P-gp structure in the closed conformation (6) (NBDs close together that was based on the bacterial Sav1866 crystal structure (7)) or the recent crystal structure of mouse P-gp in the open conformation (NBDs far apart) (8). There have been concerns that the mouse P-gp structure may be a non-native structure or in a conformation that exists very transiently (9).Our approach to map the drug translocation pathway has been to use arginine-scanning mutagenesis of the TM segments of a P-gp processing mutant (G251V) that shows partial maturation (∼15% maturation efficiency) (10). Maturation efficiency can be used to detect folding of P-gp in whole cells by monitoring the conversion of P-gp from a core-glycosylated (150 kDa) protein to a mature protein (170 kDa) that contains complex carbohydrate. Because mutant G251V shows partial maturation, we can detect whether an introduced arginine promotes, inhibits, or has a neutral effect on folding. The rationale for using arginine-scanning mutagenesis was that arginine has a large free energy barrier (17 kcal/mol) for insertion into the lipid bilayer because it is highly charged (11). Therefore, introduction of an arginine into a lipid face of the G251V mutant would likely inhibit maturation, whereas an arginine introduced into the aqueous face of the drug translocation pathway would not inhibit maturation of the mutant P-gp.In an initial study on TM1, we demonstrated the feasibility of the approach (10). All arginines introduced into the predicted lipid-facing positions inhibited maturation, whereas those introduced into positions predicted to face the drug translocation pathway did not. A particularly intriguing observation was that some arginines promoted maturation. The residues at these positions were coincidentally at positions identical to those that reacted with thiol-reactive drug substrates in cysteine-scanning mutagenesis studies and were found to be within the drug-binding pocket (10, 12). This suggested that arginine-scanning mutagenesis could be a useful approach for identifying residues in the drug translocation pathway and for determining the orientation of the TM segments in the membrane.Arginines that promote maturation appear to identify positions that are important for P-gp-drug interactions because they appear to mimic drug rescue of P-gp. It was also found that the ability of arginines (such as I306R in TM5) to promote maturation involved global enhancement of P-gp folding rather than simply compensating for a localized mutation (such as G251V) because other processing mutants could also be rescued (12). Because these arginine mutations enhance folding of P-gp in general, they will be described as enhancer rather than suppressor arginines. In this study we performed arginine-scanning mutagenesis on TMs 2–12 of P-gp processing mutant G251V to determine their orientations in the membrane and to identify residues that line the drug translocation pathway.     

Intersectin Regulates Dendritic Spine Development and Somatodendritic Endocytosis but Not Synaptic Vesicle Recycling in Hippocampal Neurons

Intersectin-short (intersectin-s) is a multimodule scaffolding protein functioning in constitutive and regulated forms of endocytosis in non-neuronal cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of Drosophila and Caenorhabditis elegans. In vertebrates, alternative splicing generates a second isoform, intersectin-long (intersectin-l), that contains additional modular domains providing a guanine nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is expressed in multiple tissues and cells, including glia, but excluded from neurons, whereas intersectin-l is a neuron-specific isoform. Thus, intersectin-I may regulate multiple forms of endocytosis in mammalian neurons, including SV endocytosis. We now report, however, that intersectin-l is localized to somatodendritic regions of cultured hippocampal neurons, with some juxtanuclear accumulation, but is excluded from synaptophysin-labeled axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV recycling. Instead intersectin-l co-localizes with clathrin heavy chain and adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces the rate of transferrin endocytosis. The protein also co-localizes with F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation during development. Our data indicate that intersectin-l is indeed an important regulator of constitutive endocytosis and neuronal development but that it is not a prominent player in the regulated endocytosis of SVs.Clathrin-mediated endocytosis (CME)⁴ is a major mechanism by which cells take up nutrients, control the surface levels of multiple proteins, including ion channels and transporters, and regulate the coupling of signaling receptors to downstream signaling cascades (1-5). In neurons, CME takes on additional specialized roles; it is an important process regulating synaptic vesicle (SV) availability through endocytosis and recycling of SV membranes (6, 7), it shapes synaptic plasticity (8-10), and it is crucial in maintaining synaptic membranes and membrane structure (11).Numerous endocytic accessory proteins participate in CME, interacting with each other and with core components of the endocytic machinery such as clathrin heavy chain (CHC) and adaptor protein-2 (AP-2) through specific modules and peptide motifs (12). One such module is the Eps15 homology domain that binds to proteins bearing NPF motifs (13, 14). Another is the Src homology 3 (SH3) domain, which binds to proline-rich domains in protein partners (15). Intersectin is a multimodule scaffolding protein that interacts with a wide range of proteins, including several involved in CME (16). Intersectin has two N-terminal Eps15 homology domains that are responsible for binding to epsin, SCAMP1, and numb (17-19), a central coil-coiled domain that interacts with Eps15 and SNAP-23 and -25 (17, 20, 21), and five SH3 domains in its C-terminal region that interact with multiple proline-rich domain proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS (16, 22-25). The rich binding capability of intersectin has linked it to various functions from CME (17, 26, 27) and signaling (22, 28, 29) to mitogenesis (30, 31) and regulation of the actin cytoskeleton (23).Intersectin functions in SV recycling at the neuromuscular junction of Drosophila and C. elegans where it acts as a scaffold, regulating the synaptic levels of endocytic accessory proteins (21, 32-34). In vertebrates, the intersectin gene is subject to alternative splicing, and a longer isoform (intersectin-l) is generated that is expressed exclusively in neurons (26, 28, 35, 36). This isoform has all the binding modules of its short (intersectin-s) counterpart but also has additional domains: a DH and a PH domain that provide guanine nucleotide exchange factor (GEF) activity specific for Cdc42 (23, 37) and a C2 domain at the C terminus. Through its GEF activity and binding to actin regulatory proteins, including N-WASP, intersectin-l has been implicated in actin regulation and the development of dendritic spines (19, 23, 24). In addition, because the rest of the binding modules are shared between intersectin-s and -l, it is generally thought that the two intersectin isoforms have the same endocytic functions. In particular, given the well defined role for the invertebrate orthologs of intersectin-s in SV endocytosis, it is thought that intersectin-l performs this role in mammalian neurons, which lack intersectin-s. Defining the complement of intersectin functional activities in mammalian neurons is particularly relevant given that the protein is involved in the pathophysiology of Down syndrome (DS). Specifically, the intersectin gene is localized on chromosome 21q22.2 and is overexpressed in DS brains (38). Interestingly, alterations in endosomal pathways are a hallmark of DS neurons and neurons from the partial trisomy 16 mouse, Ts65Dn, a model for DS (39, 40). Thus, an endocytic trafficking defect may contribute to the DS disease process.Here, the functional roles of intersectin-l were studied in cultured hippocampal neurons. We find that intersectin-l is localized to the somatodendritic regions of neurons, where it co-localizes with CHC and AP-2 and regulates the uptake of transferrin. Intersectin-l also co-localizes with actin at dendritic spines and disrupting intersectin-l function alters dendritic spine development. In contrast, intersectin-l is absent from presynaptic terminals and has little or no role in SV recycling.     

Identification of COS markers in the Pinaceae

Conserved ortholog set (COS) markers are evolutionary conserved, single-copy genes, identified from large databases of express sequence tags (ESTs). They are of particular use for constructing syntenic genetic maps among species. In this study, we identified a set of 1,813 putative single-copy COS markers between spruce and loblolly pine, then designed primers for 931 of these markers and tested these primers with DNA from spruce, pine, and Douglas fir. Of these 931 primers, 56% (524) amplified a product in both spruce and pine, and 71% (373) of these were single-banded; 224 amplicons were single-banded in all three species. Even though these COS markers were selected from large EST databases, a substantial proportion (20–30%) of amplicons displayed multiple bands or smears, suggesting significant paralogy. Sequencing of three single-banded amplicons showed high nucleotide similarities among 29 conifer species, suggesting orthology of single-banded amplicons. Screening for COS marker polymorphism in two pedigrees of white spruce and two pedigrees of loblolly pine revealed an average informativeness of 36% for spruce and 24% for pine (e.g., at least one parent was heterozygous for a single-nucleotide polymorphism within the entire amplified product). This corresponds to an average nucleotide heterozygosity of 0.05% and 0.03%, respectively, which is considerably lower than that found in other studies of spruce and pine. Thus, the advantages of COS markers for constructing syntenic maps are offset by their lower polymorphism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of the role of COP9 Signalosome (CSN) subunits in K562; the first link between CSN and autophagy

Background  

The COP9/signalosome (CSN) is a highly conserved eight subunit complex that, by deneddylating cullins in cullin-based E3 ubiquitin ligases, regulates protein degradation. Although studied in model human cell lines such as HeLa, very little is known about the role of the CSN in haemopoietic cells.     

Bayesian inference of biochemical kinetic parameters using the linear noise approximation

Background  

Fluorescent and luminescent gene reporters allow us to dynamically quantify changes in molecular species concentration over time on the single cell level. The mathematical modeling of their interaction through multivariate dynamical models requires the deveopment of effective statistical methods to calibrate such models against available data. Given the prevalence of stochasticity and noise in biochemical systems inference for stochastic models is of special interest. In this paper we present a simple and computationally efficient algorithm for the estimation of biochemical kinetic parameters from gene reporter data.     

Design and synthesis of substituted imidazole and triazole N-phenylbenzo[d]oxazolamine inhibitors of retinoic acid metabolizing enzyme CYP26

The design of N-phenylbenzo[d]oxazolamines as CYP26A1 inhibitors involved ligand docking experiments using molecular modeling (FlexX) and analysis of ligand interactions at the binding domain. The synthesis of the benzooxazol-2-yl-[phenyl-imidazol-1-yl-methyl)phenyl]amines was achieved by cyclisation of the corresponding isothiocyanates with subsequent introduction of the haem-binding heterocycle. Triazole and tetrazole derivatives were also prepared for comparison with the lead imidazole derivative. The benzooxazol-2-yl-[phenyl-imidazol-1-yl-methyl)phenyl]amines with small substituents in the phenyl ring were moderately potent CYP26A1 inhibitors (IC(50) 8 and 12 microM) and comparable with liarozole (IC(50) 7 microM).     

Isolation of 11 microsatellite markers in Crepidula convexa (Gastropoda, Calyptraeideae) for parentage analyses

Crepidula convexa, a calyptreid gastropod with direct embryonic development, changes sex from male to female in the course of its lifetime (protandry). Under sex-allocation theory, male reproductive success should be independent from age and size (a proxy used for age). However, this may be counterbalanced by female cryptic choice or gregarious behaviour. Eleven polymorphic microsatellite loci were thus developed to examine paternity of embryos and larvae. This set of loci appears suitable to carry out paternity analyses due to the high exclusion probability of unrelated males given the maternal genotype.