Biochemical characterization of distinct regions of SPEC molecules and their role in phagocytosis

Cdc42 signaling pathways play important roles in immune cell polarization and cytoskeletal changes. Although the small Cdc42-binding proteins SPEC1 and SPEC2 play a role in F-actin accumulation in activated T lymphocytes, little is known about their precise activities in other cell types. Here, we mapped the Cdc42-binding activity of SPEC1 to the CRIB sequence and a downstream alpha helical region. Biochemical studies revealed that SPEC1 did not interact with a Rac1 switch-of-function mutant capable of inducing Cdc42-like filopodia, potentially eliminating a role for SPECs in this process. A phosphoinositide-binding region was identified within a basic region N-terminal to the CRIB sequence of SPEC1. Using an anti-SPEC2 antibody, we found that endogenous SPEC2 colocalized with Cdc42 at the phagocytic cup of macrophages internalizing zymosan A particles prior to significant F-actin accumulation. Overexpression studies of the related SPEC1 protein induced marked macrophage contraction and prevented particle binding and phagocytosis. Although a Cdc42-binding mutant of SPEC1 still caused macrophage contraction, mutations within the N-terminal cysteines and phosphoinositide-binding region reversed macrophage contraction but still resulted in impaired phagocytosis. These results identify three distinct structural and functional regions within SPECs and demonstrate their likely role in early contractile events in phagocytosis.     

Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution

Microvilli are found on the surface of many cell types, including the mammalian oocyte, where they are thought to act in initial contact of sperm and oocyte plasma membranes. CD9 is currently the only oocyte protein known to be required for sperm-oocyte fusion. We found CD9 is localized to the oocyte microvillar membrane using transmission electron microscopy (TEM). Scanning electron microscopy (SEM) showed that CD9 null oocytes, which are unable to fuse with sperm, have an altered length, thickness and density of their microvilli. One aspect of this change in morphology was quantified using TEM by measuring the radius of curvature at the microvillar tips. A small radius of curvature is thought to promote fusibility and the radius of curvature of microvillar tips on CD9 wild-type oocytes was found to be half that of the CD9 null oocytes. We found that oocyte CD9 co-immunoprecipitates with two Ig superfamily cis partners, EWI-2 and EWI-F, which could have a role in linking CD9 to the oocyte microvillar actin core. We also examined latrunculin B-treated oocytes, which are known to have reduced fusion ability, and found altered microvillar morphology by SEM and TEM. Our data suggest that microvilli may participate in sperm-oocyte fusion. Microvilli could act as a platform to concentrate adhesion/fusion proteins and/or provide a membrane protrusion with a low radius of curvature. They may also have a dynamic interaction with the sperm that serves to capture the sperm cell and bring it into close contact with the oocyte plasma membrane.     

Zebrafish short fin mutations in connexin43 lead to aberrant gap junctional intercellular communication

Mutations in the zebrafish connexin43 (cx43) gene cause the short fin phenotype, indicating that direct cell-cell communication contributes to bone length. Three independently generated cx43 alleles exhibit short segments of variable sizes, suggesting that gap junctional intercellular communication may regulate bone growth. Dye coupling assays showed that all alleles are capable of forming gap junction channels. However, ionic coupling assays revealed allele-specific differences in coupling efficiency and gating. For instance, oocyte pairs expressing the weakest allele exhibited much higher levels of coupling than either of the strong alleles. Therefore, measurable differences in Cx43 function may be correlated with the severity of defects in bone length.     

CD36 signals to the actin cytoskeleton and regulates microglial migration via a p130Cas complex

The pattern recognition receptor CD36 initiates a signaling cascade that promotes microglial activation and recruitment to beta-amyloid deposits in the brain. In the present study we identify the focal adhesion-associated proteins p130Cas, Pyk2, and paxillin as novel members of the tyrosine kinase signaling pathway downstream of CD36 and show that assembly of this complex is essential for microglial migration. In primary microglia and macrophages exposed to beta-amyloid, the scaffolding protein p130Cas is rapidly tyrosine-phosphorylated and co-localizes with CD36 to membrane ruffles contemporaneous with F-actin polymerization. These beta-amyloid-stimulated events are not detected in CD36 null cells and are dependent on CD36 activation of Src family tyrosine kinases. Fyn, a Src kinase known to interact with CD36, co-precipitates with p130Cas and is an essential upstream intermediate in the signaling pathways leading to phosphorylation of the p130Cas substrate domain. Furthermore, the p130Cas-interacting kinase Pyk2 and the cytoskeletal adapter protein paxillin also demonstrate CD36-dependent phosphorylation, identifying these focal adhesion molecules as additional members of this beta-amyloid signaling cascade. Disruption of this p130Cas complex by small interfering RNA silencing inhibits p44/42 mitogen-activated protein kinase phosphorylation and microglial migration, illustrating the importance of this pathway in microglial activation and recruitment. Together, these data are the first to identify the signaling cascade that directly links CD36 to the actin cytoskeleton and, thus, implicates it in diverse processes such as cellular migration, adhesion, and phagocytosis.     

Conformation-specific binding of alpha-synuclein to novel protein partners detected by phage display and NMR spectroscopy

Alpha-synuclein (AS) is an intrinsically unstructured protein in aqueous solution but is capable of forming beta-sheet-rich fibrils that accumulate as intracytoplasmic inclusions in Parkinson disease and certain other neurological disorders. However, AS binding to phospholipid membranes leads to a distinct change in protein conformation, stabilizing an extended amphipathic alpha-helical domain reminiscent of the exchangeable apolipoproteins. To better understand the significance of this conformational change, we devised a novel bacteriophage display screen to identify protein binding partners of helical AS and have identified 20 proteins with roles in diverse cellular processes related to membrane trafficking, ion channel modulation, redox metabolism, and gene regulation. To verify that the screen identifies proteins with specificity for helical AS, we further characterized one of these candidates, endosulfine alpha (ENSA), a small cAMP-regulated phosphoprotein implicated in the regulation of insulin secretion but also expressed abundantly in the brain. We used solution NMR to probe the interaction between ENSA and AS on the surface of SDS micelles. Chemical shift perturbation mapping experiments indicate that ENSA interacts specifically with residues in the N-terminal helical domain of AS in the presence of SDS but not in aqueous buffer lacking SDS. The ENSA-related protein ARPP-19 (cAMP-regulated phosphoprotein 19) also displays specific interactions with helical AS. These results confirm that the helical N terminus of AS can mediate specific interactions with other proteins and suggest that membrane binding may regulate the physiological activity of AS in vivo.     

Arabidopsis VTC2 encodes a GDP-L-galactose phosphorylase, the last unknown enzyme in the Smirnoff-Wheeler pathway to ascorbic acid in plants

The first committed step in the biosynthesis of L-ascorbate from D-glucose in plants requires conversion of GDP-L-galactose to L-galactose 1-phosphate by a previously unidentified enzyme. Here we show that the protein encoded by VTC2, a gene mutated in vitamin C-deficient Arabidopsis thaliana strains, is a member of the GalT/Apa1 branch of the histidine triad protein superfamily that catalyzes the conversion of GDP-L-galactose to L-galactose 1-phosphate in a reaction that consumes inorganic phosphate and produces GDP. In characterizing recombinant VTC2 from A. thaliana as a specific GDP-L-galactose/GDP-D-glucose phosphorylase, we conclude that enzymes catalyzing each of the ten steps of the Smirnoff-Wheeler pathway from glucose to ascorbate have been identified. Finally, we identify VTC2 homologs in plants, invertebrates, and vertebrates, suggesting that a similar reaction is used widely in nature.     

Peroxisome proliferator-activated receptor gamma 2 and acyl-CoA synthetase 5 polymorphisms influence diet response

Peroxisome proliferator-activated receptor gamma (PPARgamma) and its response gene, Acyl CoA synthetase 5 (ACSL5), which has an important role in fatty acid metabolism, may affect weight loss in response to caloric restriction. Therefore, we aimed to determine whether these genes were involved in the interindividual response to dietary treatment. Genotypic/phenotypic comparisons were made between selected obese women from the quintiles losing the most (diet responsive, n = 74) and the quintiles losing the least (diet-resistant, n = 67) weight in the first 6 weeks of a 900-kcal formula diet. Two common PPARgamma single nucleotide polymorphisms, Pro(12)Ala and C1431T, and eight polymorphisms across the ACSL5 gene were selected for single locus and haplotypic association analyses. The PPARgamma Pro(12)Ala single nucleotide polymorphism was associated with diet resistance (odds ratio = 3.48, 95% confidence interval = 1.41 to 8.56, p = 0.03), and the rs2419621, located in the 5'untranslated region of the ACSL5 gene, displayed the strongest association with diet response (odds ratio = 3.45, 95% confidence interval = 1.61 to 7.69, p = 0.001). Skeletal muscle ACSL5 mRNA expression was significantly lower in carriers of the wildtype compared with the variant rs2419621 allele (p = 0.03). Our results suggest a link between PPARgamma2 and ACSL5 genotype and diet responsiveness.     

Controlled study of critical parent and family factors in the obesigenic environment

Objective: Critical gaps remain in our understanding of the obesigenic family environment. This study examines parent and family characteristics among obese youth presenting for treatment in a clinic setting. Research Methods and Procedures: Families of 78 obese youth (BMI z‐score = 2.4; age, 8 to 16 years; 59% girls; 49% African‐American) were compared with 71 non‐overweight (BMI z‐score = ?0.02) demographically matched comparisons. Parents completed measures assessing family demographics, psychological distress (Symptom Checklist 90‐Revised), and family functioning both broadly (Family Environment Scale: Conflicted, Support, Control) and at mealtimes (About Your Child's Eating‐Revised: Mealtime Challenges, Positive Mealtime Interaction). Height and weight were obtained from all participants. Results: Compared with mothers and fathers of non‐overweight youth, parents of obese youth had significantly higher BMIs (p < 0.001). Mothers of obese youth reported significantly greater psychological distress (p < 0.01), higher family conflict (p < 0.05), and more mealtime challenges (p < 0.01). Less positive family mealtime interactions were reported by both mothers (p < 0.01) and fathers (p < 0.05) of obese youth. These group differences did not vary by child sex or race. Logistic regression analyses indicated that maternal distress and mealtime challenges discriminated between obese and non‐overweight youth after controlling for maternal BMI. Family conflict was explained, in part, by maternal distress. Discussion: Obese youth who present for treatment in a clinic setting are characterized by psychosocial factors at the parent and family level that differ from non‐overweight youth. These data are critical because they identify factors that may be serving as barriers to a family's or youth's ability to implement healthy lifestyle behaviors but that are potentially modifiable.     

Campylobacter jejuni: molecular biology and pathogenesis

Campylobacter jejuni is a foodborne bacterial pathogen that is common in the developed world. However, we know less about its biology and pathogenicity than we do about other less prevalent pathogens. Interest in C. jejuni has increased in recent years as a result of the growing appreciation of its importance as a pathogen and the availability of new model systems and genetic and genomic technologies. C. jejuni establishes persistent, benign infections in chickens and is rapidly cleared by many strains of laboratory mouse, but causes significant inflammation and enteritis in humans. Comparing the different host responses to C. jejuni colonization should increase our understanding of this organism.