Cbl-mediated ubiquitinylation is required for lysosomal sorting of epidermal growth factor receptor but is dispensable for endocytosis

Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identified Cbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl-/- mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization.     

The Gla domain of factor IXa binds to factor VIIIa in the tenase complex

During blood coagulation factor IXa binds to factor VIIIa on phospholipid membranes to form an enzymatic complex, the tenase complex. To test whether there is a protein-protein contact site between the gamma-carboxyglutamic acid (Gla) domain of factor IXa and factor VIIIa, we demonstrated that an antibody to the Gla domain of factor IXa inhibited factor VIIIa-dependent factor IXa activity, suggesting an interaction of the factor IXa Gla domain with factor VIIIa. To study this interaction, we synthesized three analogs of the factor IXa Gla domain (FIX1-47) with Phe-9, Phe-25, or Val-46 replaced, respectively, with benzoylphenylalanine (BPA), a photoactivatable cross-linking reagent. These factor IX Gla domain analogs maintain native tertiary structure, as demonstrated by calcium-induced fluorescence quenching and phospholipid binding studies. In the absence of phospholipid membranes, FIX1-47 was able to inhibit factor IXa activity. This inhibition is dependent on the presence of factor VIIIa, suggesting a contact site between the factor IXa Gla domain and factor VIIIa. To demonstrate a direct interaction we did cross-linking experiments with FIX1-479BPA, FIX1-4725BPA, and FIX1-4746BPA. Covalent cross-linking to factor VIIIa was observed primarily with FIX1-4725BPA and to a much lesser degree with FIX1-4746BPA. Immunoprecipitation experiments with an antibody to the C2 domain of factor VIIIa indicate that the factor IX Gla domain cross-links to the A3-C1-C2 domain of factor VIIIa. These results suggest that the factor IXa Gla domain contacts factor VIIIa in the tenase complex through a contact site that includes phenylalanine 25 and perhaps valine 46.     

Highly conserved cysteines of mouse core 2 beta1,6-N-acetylglucosaminyltransferase I form a network of disulfide bonds and include a thiol that affects enzyme activity

Core 2 beta1,6-N-acetylglucosaminyltransferase I (C2GnT-I) plays a pivotal role in the biosynthesis of mucin-type O-glycans that serve as ligands in cell adhesion. To elucidate the three-dimensional structure of the enzyme for use in computer-aided design of therapeutically relevant enzyme inhibitors, we investigated the participation of cysteine residues in disulfide linkages in a purified murine recombinant enzyme. The pattern of free and disulfide-bonded Cys residues was determined by liquid chromatography/electrospray ionization tandem mass spectrometry in the absence and presence of dithiothreitol. Of nine highly conserved Cys residues, under both conditions, one (Cys217) is a free thiol, and eight are engaged in disulfide bonds, with pairs formed between Cys59-Cys413, Cys100-Cys172, Cys151-Cys199, and Cys372-Cys381. The only non-conserved residue within the beta1,6-N-acetylglucosaminyltransferase family, Cys235, is also a free thiol in the presence of dithiothreitol; however, in the absence of reductant, Cys235 forms an intermolecular disulfide linkage. Biochemical studies performed with thiolreactive agents demonstrated that at least one free cysteine affects enzyme activity and is proximal to the UDP-GlcNAc binding site. A Cys217 --> Ser mutant enzyme was insensitive to thiol reactants and displayed kinetic properties virtually identical to those of the wild-type enzyme, thereby showing that Cys217, although not required for activity per se, represents the only thiol that causes enzyme inactivation when modified. Based on the pattern of free and disulfide-linked Cys residues, and a method of fold recognition/threading and homology modeling, we have computed a three-dimensional model for this enzyme that was refined using the T4 bacteriophage beta-glucosyltransferase fold.     

Reduced resistance of invasive varieties of the alien tree<Emphasis Type="Italic"> Sapium sebiferum</Emphasis> to a generalist herbivore

Invasive plants are often larger in their introduced range compared to their native range. This may reflect an evolved reduction in defense and increase in growth in response to low herbivory in their introduced range. Key elements of this scenario include genetic differences in defense and growth yet uniformly low rates of herbivory in the field that dissociate defense and herbivore damage for alien species. We conducted a laboratory experiment with Melanoplus angustipennis grasshoppers and Chinese Tallow Tree seedlings ( Sapium sebiferum) from its native range (China) and its introduced range (Texas, USA) where it is invasive. We caged grasshoppers with pairs of Sapium seedlings from the same continent or different continents. The amounts of leaf area removed from Texas and China seedlings, and their height growth rates, were indistinguishable when both seedlings in the pair were from the same continent. However, when grasshoppers had a choice between seedlings from different continents, they removed more Texas Sapium foliage than China Sapium foliage and height growth rates were higher for China Sapium seedlings compared to Texas seedlings. Grasshopper growth rates increased with greater Sapium foliage consumption. In a common garden in Texas, Sapium seedlings from Texas grew 40% faster than those from China. Chewing insect herbivores removed little Sapium foliage in the field experiment. Although grasshoppers preferred to feed on Texas Sapium when offered a choice in the laboratory, extremely low herbivory levels in the field may have allowed the Texas seedlings to outperform the China seedlings in the common garden. These results demonstrate post-invasion genetic differences in herbivore resistance and growth of an invasive plant species together with a decoupling of defense and herbivore choice in the introduced range.     

Mussel disturbance dynamics: signatures of oceanographic forcing from local interactions

Local interactions, biotic and abiotic, can have a strong influence on the large-scale properties of ecosystems. However, ecological models often explore the influence of local biotic interactions where physical disturbance is included as a large-scale and imposed source of variability but is not allowed to interact with biotic processes at the local scale. In marine intertidal communities dominated by mussels, wave disturbances create gaps in the mussel bed that recover through a successional sequence. We present a lattice model of mussel disturbance dynamics that allows local interactions between wave disturbance and mussel recolonization, in which each cell of the lattice can be empty, occupied by a mussel bed element, or disturbed (which corresponds to a newly disturbed cell that has unstable edges). As in natural ecosystems, wave disturbance can also spread from disturbed to adjacent occupied cells, and recolonization can also spread from occupied to adjacent empty cells. We first validate the local rules from artificial gap experiments and from natural gap monitoring along the Oregon coast. We analyze the properties of the model system as a function of different oceanographic forcings of productivity and disturbance. We show that the mussel bed can go through phase transitions characterized by a large sensitivity of mussel cover and patterns to oceanographic forcings but also that criticality (scale invariance) is observed over wide ranges of parameters, which suggests self-organization. We also show that spatial patterns in the intertidal can provide a robust signature of local processes and can inform about oceanographic regimes. We do so by comparing the large-scale patterns of the simulation (scaling exponents) with field data, which suggest that some experimental sites are close to criticality. Our results suggest that regional patterns in disturbed populations can be explained by local biotic and abiotic processes submitted to oceanographic forcing.     

Extraction and reconstitution of calponin and consequent contractile ability in permeabilized smooth muscle fibers

We demonstrate reduction and restoration of contractile ability in response to protein extraction and reconstitution in Triton X-100/glycerol-permeabilized smooth muscle fibers. Through significant reduction in the content of caldesmon (CaD), calponin (CaP), and the 20-kDa regulatory light chain (RLC) of myosin, but not other contractile proteins in "chemically skinned" fibers, we substantially reduced the contractile ability of these fibers, as measured by their ability to generate isometric force and to hydrolyze ATP by actomyosin Mg2+ ATPase. When the protein-depleted fibers were then reconstituted (either with a mixture of purified protein standards of CaD, CaP, and myosin RLC or with a protein extract from the demembranized muscle fibers containing CaD, CaP, and myosin RLC plus several low-molecular-mass proteins), all proteins used for reincorporation returned nearly to control levels, as did isometric force generation and rate of ATP hydrolysis. The fact that the low-molecular-mass proteins do not affect contractility in this model system indicates that our methods for reversible modulation of the content of CaP and CaD may provide a valuable tool for studying the thin-filament-based regulation of contractility.     

Oestrous state dynamics in chemical communication by captive female Asian elephants

In many mammals, reproductive status is revealed through chemical cues in urine. The reproductive status of receivers may influence their interest in such signals. For social mammals that live in matrilineal groups, females may benefit by detecting the reproductive condition of herdmates. Responses to urine during oestrous cycles of senders and receivers are potential indicators of signal functions. We examined the chemosensory responses, first by four captive female Asian elephants,Elephas maximus , over their oestrous cycles to familiar follicular and luteal phase urine and second by 14 different female Asian elephants to unfamiliar conspecific follicular and luteal phase urine. We asked whether females could distinguish the reproductive state of another female as measured by their differential response to luteal- and follicular-phase urine. We further examined the influence of the receiver's reproductive status on response levels. Females responded more with specific tactolfactory trunk behaviours to follicular- than to luteal-phase urine, but only when the receiving female was in her follicular phase. Like their male conspecifics, Asian elephant females can detect changes in the reproductive state of conspecifics. The functional significance of this ability has yet to be determined but may be related more to the resource holding power of females in follicular phase than to a means for females to synchronize oestrous cycles. Such female-female communication may have important effects on social group dynamics. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour.      

A role for apolipoprotein(a) in protection of the low-density lipoprotein component of lipoprotein(a) from copper-mediated oxidation

Low-density lipoprotein (LDL) oxidation is stimulated by copper. Addition of a recombinant form of apolipoprotein(a) (apo(a); the distinguishing protein component of lipoprotein(a)) containing 17 plasminogen kringle IV-like domains (17K r-apo(a)) protects LDL against oxidation by copper. Protection is specific to apo(a) and is not achieved by plasminogen or serum albumin. When Cu(2+) is added to 17K r-apo(a), its intrinsic fluorescence is quenched in a concentration-dependent and saturable manner. Quenching is unchanged whether performed aerobically or anaerobically and is reversible by ethylenediaminetetraacetate, suggesting that it is due to equilibrium binding of Cu(2+) and not to oxidative destruction of tryptophan residues. The fluorescence change exhibits a sigmoid dependence on copper concentration, and time courses of quenching are complex. At copper concentrations below 10 microM there is little quenching, whereas above 10 microM quenching proceeds immediately as a double-exponential decay. The affinity and kinetics of copper binding to 17K r-apo(a) are diminished in the presence of the lysine analogue epsilon -aminocaproic acid. We propose that copper binding to the kringle domains of 17K is mediated by a His-X-His sequence that is located about 5A from the closest tryptophan residue of the lysine binding pocket. Copper binding may account for the natural resistance to copper-mediated oxidation of lipoprotein(a) relative to LDL that has been previously reported and for the protection afforded by apo(a) from copper-mediated oxidation of LDL that we describe in the present study.