Hermansky-Pudlak Syndrome Protein Complexes Associate with Phosphatidylinositol 4-Kinase Type II �� in Neuronal and Non-neuronal Cells

The Hermansky-Pudlak syndrome is a disorder affecting endosome sorting.Disease is triggered by defects in any of 15 mouse gene products, which arepart of five distinct cytosolic molecular complexes: AP-3, homotypic fusionand vacuole protein sorting, and BLOC-1, -2, and -3. To identify molecularassociations of these complexes, we used in vivo cross-linkingfollowed by purification of cross-linked AP-3 complexes and mass spectrometricidentification of associated proteins. AP-3 was co-isolated with BLOC-1,BLOC-2, and homotypic fusion and vacuole protein sorting complex subunits;clathrin; and phosphatidylinositol-4-kinase type II α (PI4KIIα).We previously reported that this membrane-anchored enzyme is a regulator ofAP-3 recruitment to membranes and a cargo of AP-3 (Craige, B.,Salazar, G., and Faundez, V. (2008) Mol. Biol.Cell19,1415-1426). Using cells deficientin different Hermansky-Pudlak syndrome complexes, we identified that BLOC-1,but not BLOC-2 or BLOC-3, deficiencies affect PI4KIIα inclusion intoAP-3 complexes. BLOC-1, PI4KIIα, and AP-3 belong to a tripartitecomplex, and down-regulation of either PI4KIIα, BLOC-1, or AP-3complexes led to similar LAMP1 phenotypes. Our analysis indicates that BLOC-1complex modulates the association of PI4KIIα with AP-3. These resultssuggest that AP-3 and BLOC-1 act, either in concert or sequentially, tospecify sorting of PI4KIIα along the endocytic route.Membranous organelles along the exocytic and endocytic pathways are eachdefined by unique lipid and protein composition. Vesicle carriers communicateand maintain the composition of these organelles(2). Consequently defining themachineries that specify vesicle formation, composition, and delivery arecentral to understanding membrane protein traffic. Generally vesiclebiogenesis uses multiprotein cytosolic machineries to select membranecomponents for inclusion in nascent vesicles(2,3). Heterotetrameric adaptorcomplexes (AP-1 to AP-4) are critical to generate vesicles of specificcomposition from the different organelles constituting the exocytic andendocytic routes(2-4).The best understood vesicle formation machinery in mammalian cells is theone organized around the adaptor complex AP-2(5). This complex generatesvesicles from the plasma membrane using clathrin. Our present detailedunderstanding of AP-2 vesicle biogenesis mechanisms and interactions emergedfrom a combination of organellar and in vitro binding proteomicsanalyses together with the study of binary interactions in cell-free systems(5-9).In contrast, the vesicle biogenesis pathways controlled by AP-3 are far lessunderstood. AP-3 functions to produce vesicles that traffic selected membraneproteins from endosomes to lysosomes, lysosome-related organelles, or synapticvesicles(10-13).AP-3 is one of the protein complexes affected in the Hermansky-Pudlak syndrome(HPS;³ OnlineMendelian Inheritance in Man (OMIM) 203300). So far, mutations in any of 15mouse or eight human genes trigger a common syndrome. This syndromeencompasses defects that include pigment dilution, platelet dysfunction,pulmonary fibrosis, and occasionally neurological phenotypes(14,15). All forms of HPS showdefective vesicular biogenesis or trafficking that affects lysosomes,lysosome-related organelles (for example melanosomes and platelet densegranules), and, in some of them, synaptic vesicles(11-13).Most of the 15 HPS loci encode polypeptides that assemble into five distinctmolecular complexes: the adaptor complex AP-3, HOPS, and the BLOC complexes 1,2, and 3 (14). Recently binaryinteractions between AP-3 and BLOC-1 or BLOC-1 and BLOC-2 suggested thatarrangements of these complexes could regulate membrane protein targeting(16). Despite the abundance ofgenetic deficiencies leading to HPS and genetic evidence that HPS complexesmay act on the same pathway in defined cell types(17), we have only a partialpicture of protein interactions organizing these complexes and how they mightcontrol membrane protein targeting.In this study, we took advantage of cell-permeant and reversiblecross-linking of HPS complexes followed by their immunoaffinity purificationto identify novel molecular interactions. Cross-linked AP-3 co-purified withBLOC-1, BLOC-2, HOPS, clathrin, and the membrane protein PI4KIIα. Wepreviously identified PI4KIIα as a cargo and regulator of AP-3recruitment to endosomes (1,18). Using mutant cellsdeficient in either individual HPS complexes or a combination of them, wefound that BLOC-1 facilitates the interaction of AP-3 and PI4KIIα. Ourstudies demonstrate that subunits of four of the five HPS complexes co-isolatewith AP-3. Moreover BLOC-1, PI4KIIα, and AP-3 form a tripartite complexas demonstrated by sequential co-immunoprecipitations as well as by similarLAMP1 distribution phenotypes induced by down-regulation of components of thistripartite complex. Our findings indicate that BLOC-1 complex modulates therecognition of PI4KIIα by AP-3. These data suggest that AP-3, either inconcert or sequentially with BLOC-1, participates in the sorting of commonmembrane proteins along the endocytic route.     

Coupled Diffusion of Peripherally Bound Peptides along the Outer and Inner Membrane Leaflets

Transmembrane signaling implies that peripheral protein binding to one leaflet be detected by the opposite leaflet. Therefore, protein recruitment into preexisting cholesterol and sphingolipid rich platforms may be required. However, no clear molecular picture has evolved about how these rafts in both leaflets are connected. By using planar lipid bilayers, we show that the peripheral binding of a charged molecule (poly-lysine, PLL) is detected at the other side of the bilayer without involvement of raft lipids. The diffusion coefficient, D_P, of PLL differed by a factor of √2 when PLL absorbed to one or to both leaflets of planar membranes. Fluorescence correlation spectroscopy showed that the changes of the lipid diffusion coefficient, D_M, were even more pronounced. Although D_M remained larger than D_P on PLL binding to the first membrane leaflet, D_M dropped to D_P on PLL binding to both leaflets, which indicated that the lipids sandwiched between two PLL molecules had formed a nanodomain. Due to its small area of ∼20 nm² membrane electrostriction or leaflet interaction at bilayer midplane can only make a small contribution to interleaflet coupling. The tendency of the system to maximize the area where the membrane is free to undulate seems to be more important. As a spot with increased bending stiffness, the PLL bound patch in one leaflet attracts a stiffening additive on the other leaflet. That is to say, instead of suppressing undulations in two spots, two opposing PLL molecules migrate along a membrane at matching positions and suppress these undulations in a single spot. The gain in undulation energy is larger than the energy required for the alignment of two small PLL domains in opposite leafs and their coordinated diffusion. We propose that this type of mechanical interaction between two membrane separated ligands generally contributes to transmembrane signaling.     

Genetic and environmental heterogeneity of residual variance of weight traits in Nellore beef cattle

Background

Many studies have provided evidence of the existence of genetic heterogeneity of environmental variance, suggesting that it could be exploited to improve robustness and uniformity of livestock by selection. However, little is known about the perspectives of such a selection strategy in beef cattle.

Methods

A two-step approach was applied to study the genetic heterogeneity of residual variance of weight gain from birth to weaning and long-yearling weight in a Nellore beef cattle population. First, an animal model was fitted to the data and second, the influence of additive and environmental effects on the residual variance of these traits was investigated with different models, in which the log squared estimated residuals for each phenotypic record were analyzed using the restricted maximum likelihood method. Monte Carlo simulation was performed to assess the reliability of variance component estimates from the second step and the accuracy of estimated breeding values for residual variation.

Results

The results suggest that both genetic and environmental factors have an effect on the residual variance of weight gain from birth to weaning and long-yearling in Nellore beef cattle and that uniformity of these traits could be improved by selecting for lower residual variance, when considering a large amount of information to predict genetic merit for this criterion. Simulations suggested that using the two-step approach would lead to biased estimates of variance components, such that more adequate methods are needed to study the genetic heterogeneity of residual variance in beef cattle.     

Aerobic training increases the expression of adiponectin receptor genes in the peripheral blood mononuclear cells of young men

Little is known about the effect of exercise training on the expression of adiponectin receptor genes in peripheral blood mononuclear cells (PBMCs). In this study, we investigated the effects of aerobic training on the expression of AdipoR1 and AidpoR2 mRNAs in PBMCs, whole body insulin sensitivity, and circulating adiponectins in men. Thirty young men were randomly assigned to either a control (n=15) or an exercise (n=15) group. Subjects assigned to the exercise group underwent a 12-week jogging and/or running programme on a motor-driven treadmill at an intensity of 60%-75% of the age-based maximum heart rate with duration of 40 minutes per session and a frequency of 5 days per week. Two-way mixed ANOVA with repeated measures was used to test any significant time-by-group interaction effects for the measured variables at p=0.05. We found significant time-by-group interaction effects for waist circumference (p=0.001), VO_2max (p<0.001), fasting insulin (p=0.016), homeostasis model assessment for insulin resistance (HOMA-IR) (p=0.010), area under the curve (AUC) for insulin response during the 75-g oral glucose tolerance test (p=0.002), high-molecular weight (HMW) adiponectin (p=0.016), and the PBMC mRNA levels of AdipoR1 (p<0.001) and AdipoR2 (p=0.001). The exercise group had significantly increased mRNA levels of AdipoR1 and AdipoR2 in PBMCs, along with increased whole body insulin sensitivity and HMW adiponectin, decreased waist circumference, and increased VO_2max compared with the control group. In summary, the current findings suggest that exercise training modulates the expression of AdipoR1 and AdipoR2 mRNAs in PBMCs, implying that manipulation of the expression of these genes could be a potential surrogate for lifestyle intervention-mediated improvements of whole body insulin sensitivity and glucose homeostasis.     

Effect of carbon dioxide on differentiation and on the level of a soluble b-type cytochrome in Phycomyces blakesleeanus

A soluble b-type cytochrome has been detected and partly characterized in mycelial extracts of Phycomyces blakesleeanus. As it is already known, CO₂ delays sporangiophorogenesis, but it also lowers the level of this cytochrome. A possible causal relationship between sporangiophorogenesis and the b-type-cytochrome level may exist. There is some correlation between the extent of the delay of sporangiophorogenesis and of the decrease in cytochrome-b level in wild type and mutants that are either resistant or sensitive to CO₂.     

Highly specialized Cretaceous beetle parasitoids (Ripiphoridae) identified with optimized visualization of microstructures

Extremely miniaturized longipedes insects (body length c. 0.3 mm) embedded in two pieces of Cretaceous amber from Myanmar are described and interpreted. Using inverted fluorescence and light microscopy for detailed analysis of microstructures, the inclusions were identified as primary larvae of the beetle family Ripiphoridae, subfamily Ripidiinae. While the structure of thoracic and abdominal segments including appendages corresponds well with the groundplan known in recent members of Ripidiinae, a curved prosternal ridge with prominent spines (each c. 5 μm), the reduced condition of stemmata and antennae and the lack of sharp mandibles are unique features within the entire family, apparently apomorphies of the longipedes larvae. A sinuate prosternal edge with a dense row of spines (prosternoctenidium) might be homologous with ‘head ctenidia’ in some previously described miniaturized conicocephalate larvae, but further investigation is needed. The morphological differences between the head of longipedes larvae and extant Ripidiinae are interpreted as adaptations to different groups of hosts and life strategies. Palaeoethology of the longipedes larvae is briefly discussed. In addition, the systematic placement of conicocephalate larvae from Canadian, Myanmar and Russian Cretaceous ambers, already interpreted by various authors as primary instars within Coleopterida (assigned to either Strepsiptera or to the coleopteran Tenebrionoidea: Ripiphoridae), is discussed.     

Pitfalls of using lucigenin in endothelial cells: Implications for NAD(P)H dependent superoxide formation

Since an increased endothelial superoxide formation plays an important role in the pathogenesis of endothelial dysfunction its specific detection is of particular interest. The widely used superoxide probe lucigenin, however, has been reported to induce superoxide under certain conditions, especially in the presence of NADH. This raises questions as to the conclusion of a NAD(P)H oxidase as the major source of endothelial superoxide. Using independent methods, we showed that lucigenin in the presence of NADH leads to the production of substantial amount of superoxide (～ 15-fold of control) in endothelial cell homogenates. On the other hand, these independent methods revealed that endothelial cells without lucigenin still produce superoxide in a NAD(P)H-dependent manner. This was blocked by inhibitors of the neutrophil NADPH oxidase diphenyleniodonium and phenylarsine oxide. Our results demonstrate that a NAD(P)H-dependent oxidase is an important source for endothelial superoxide but the latter, however, cannot be measured reliably by lucigenin.     

Membrane transport of singlet oxygen monitored by dipole potential measurements

The efficiency of photodynamic reactions depends on 1), the penetration depth of the photosensitizer into the membrane and 2), the sidedness of the target. Molecules which are susceptible to singlet oxygen (¹O₂) experience less damage when separated from the photosensitizer by the membrane. Since ¹O₂ lifetime in the membrane environment is orders of magnitude longer than the time required for nonexcited oxygen (O₂) to cross the membrane, this observation suggests that differences between the permeabilities or membrane partition of ¹O₂ and O₂ exist. We investigated this hypothesis by releasing ¹O₂ at one side of a planar membrane while monitoring the kinetics of target damage at the opposite side of the same membrane. Damage to the target, represented by dipole-modifying molecules (phloretin or phlorizin), was indicated by changes in the interleaflet dipole potential difference Δφ_b. A simple analytical model allowed estimation of the ¹O₂ interleaflet concentration difference from the rate at which Δφ_b changed. It confirmed that the lower limit of ¹O₂ permeability is ∼2 cm/s; i.e., it roughly matches O₂ permeability as predicted by Overton's rule. Consequently, the membrane cannot act as a barrier to ¹O₂ diffusion. Differences in the reaction rates at the cytoplasmic and extracellular membrane leaflets may be attributed only to ¹O₂ quenchers inside the membrane.     

Syntheses of 1-[2-(Phosphonomethoxy)Alkyl]Thymine Monophosphates and an Evaluation of their Inhibitory Activity Toward Human Thymidine Phosphorylase

A series of new monophosphates of 1-[2-(phosphonomethoxy)alkyl]thymines, such as PMPTp_, 3-MeO-PMPTp, HPMPTp, and FPMPTp, were synthesized and tested for their ability to inhibit human thymidine phosphorylase. Kinetic measurements of enzyme activity were performed using thymidine and inorganic phosphate as the substrates. The data show that some monophosphates provide a considerable increase of the multisubstrate inhibitory effect. The highest inhibitory potency was found with (R)-FPMPTp 4c (K _i ^dT = 4.09 ± 0.47 μM, K _i(P_i) = 2.13 ± 0.29 μM) and (R) 3-MeO-PMPTp 4d (K _i ^dT = 5.78 ± 0.71 μM, K _i(P_i) = 2.71 ± 0.37 μM).