Epidermal growth factor contains both positive and negative determinants for interaction with ErbB-2/ErbB-3 heterodimers

Epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha are potent activators of the ErbB-1 receptor, but, unlike TGF-alpha, EGF is also a weak activator of ErbB-2/ErbB-3 heterodimers. To understand the specificity of EGF-like growth factors for binding to distinct ErbB members, we used EGF/TGF-alpha chimeras to examine the requirements for ErbB-2/ErbB-3 activation. Here we show that in contrast to these two wild-type ligands, distinct EGF/TGF-alpha chimeras are potent activators of ErbB-2/ErbB-3 heterodimers. On the basis of differences in the potency of these various chimeras, specific residues in the linear N-terminal region and the so-called B-loop of these ligands were identified to be involved in interaction with ErbB-2/ErbB-3. A chimera consisting of human EGF sequences with the linear N-terminal region of human TGF-alpha was found to be almost as potent as the natural ligand neuregulin (NRG)-1beta in activating 32D cells expressing ErbB-2/ErbB-3 and human breast cancer cells. Binding studies revealed that this chimera, designated T1E, has high affinity for ErbB-2/ErbB-3 heterodimers, but not for ErbB-3 alone. Subsequent exchange studies revealed that introduction of both His2 and Phe3 into the linear N-terminal region was already sufficient to make EGF a potent activator of ErbB-2/ErbB-3 heterodimers, indicating that these two amino acids contribute positively to this receptor binding. Analysis of the B-loop revealed that Leu26 in EGF facilitates interaction with ErbB-2/ErbB-3 heterodimers, while the equivalent Glu residue in TGF-alpha impairs binding. Since all EGF/TGF-alpha chimeras tested have maintained high binding affinity for ErbB-1, it is concluded that the diversity of the ErbB signaling network is determined by specific amino acids that facilitate binding to one receptor member, in addition to residues that impede binding to other ErbB family members.     

Temperature‐dependent conformational change affecting Tyr11 and sweetness loops of brazzein

The sweet protein brazzein, a member of the Csβα fold family, contains four disulfide bonds that lend a high degree of thermal and pH stability to its structure. Nevertheless, a variable temperature study has revealed that the protein undergoes a local, reversible conformational change between 37 and 3°C with a midpoint about 27°C that changes the orientations and side‐chain hydrogen bond partners of Tyr8 and Tyr11. To test the functional significance of this effect, we used NMR saturation transfer to investigate the interaction between brazzein and the amino terminal domain of the sweet receptor subunit T1R2; the results showed a stronger interaction at 7°C than at 37°C. Thus the low temperature conformation, which alters the orientations of two loops known to be critical for the sweetness of brazzein, may represent the bound state of brazzein in the complex with the human sweet receptor. Proteins 2013; © 2012 Wiley Periodicals, Inc.     

Apical transport of osteopontin is independent of N-glycosylation and sialylation

Studies of how epithelial surface polarity into apical and basolateral domains is generated and maintained have proposed that carbohydrate modifications serve as apical targeting signals for proteins by interacting with lectin sorters. However, the experimental evidence in support of N-glycans, O-glycans and sialic acids mediating apical transport is still very controversial. This could be partly due to the fact that in most studies exogenously expressed proteins were analysed. One has, therefore, examined the role of carbohydrate moieties in apical targeting of the endogenous secretory protein osteopontin in MDCK cells. It was found, however, that sorting of osteopontin does not require N-glycosylation of the protein itself nor that of other factors involved in the sorting process. Incubation of cells with the inhibitor of O-glycosylation benzyl- &#102 -GalNAc reduced the molecular weight of osteopontin by blocking sialic acid addition to O-glycans. Interestingly, also impairment of sialylation had no effect on polar secretion of the protein. Thus, the results show that both N-glycans and sialic acids are not essential sorting signals, suggesting that inner core carbohydrates and/or a proteinaceous signal mediate apical targeting of osteopontin.     

Anti-hypertensive effects of Rosuvastatin are associated with decreased inflammation and oxidative stress markers in hypertensive rats

Among their pleiotropic effects, statins exert antioxidant and anti-inflammatory properties. The aim of this study was to evaluate in normotensive (WKY) and in spontaneously hypertensive rats (SHR) the effect of rosuvastatin (ROSU) treatment on (1) plasma inflammation markers and endogenous NO synthase inhibitor (ADMA) levels, (2) reactive oxygen species (ROS) generated by circulating leukocytes and (3) vascular oxidative stress and tissue inflammation markers. Plasma cytokines were higher in SHR than in WKY, except for IL-4, which was lower in SHR than in WKY. SHR monocytes exhibited higher production of ROS than did WKY monocytes. In the experimental conditions, ROSU did not modify plasma cholesterol levels in SHR but attenuated the increase in systolic blood pressure. In SHR only, ROSU lessened pro-inflammatory cytokines and ADMA levels, increased IL-4 and reduced ROS production in circulating monocytes. These results demonstrate the beneficial effects of ROSU in SHR, independently of any lowering of cholesterol levels.     

Latirhinus uitstlani,a ‘broad-nosed’ saurolophine hadrosaurid (Dinosauria,Ornithopoda) from the late Campanian (Cretaceous) of northern Mexico

We describe a new genus and species of hadrosaurid dinosaur, Latirhinus uitstlani, from upper Campanian strata of the Cerro del Pueblo Formation in northern Mexico. The new taxon differs from other hadrosaurids, except Gryposaurus, in possessing a deep and arcuate dorsal process of the nasal; it differs from Gryposaurus, as well as from all other saurolophines, in having a broader narial foramen, and an anterodorsally curved deltoid crest of the scapula (convergent with Lambeosaurinae within the context of Hadrosauridae). L. uitstlani appears to be allied to ‘kritosaurs’, particularly to species of Gryposaurus, and it represents one of the southernmost saurolophines of North America. Likewise, L. uitstlani adds to the diversity of saurolophines in the southern Western Interior Basin and its occurrence in northern Mexico is consistent with the apparent predominance of ‘kritosaurs’ in southern Laramidia during late Campanian times.     

Genetic Analysis of Population Structure and Reproductive Mode of the Termite Reticulitermes chinensis Snyder

The subterranean termite Reticulitermes chinensis Snyder is an important pest of trees and buildings in China. Here, we characterized genetic structure and reproductive modes of R. chinensis from China for the first time. A total of 1,875 workers from 75 collection sites in Huanggang, Changsha and Chongqing cities were genotyped at eight microsatellite loci. Analysis of genetic clusters showed two subpopulations in Chongqing city. The Huanggang population showed a uniform genetic pattern and was separated from the other populations by the largest genetic distances (F _ST: 0.17–0.20). In contrast, smaller genetic distances (F _ST: 0.05–0.12) separated Changsha, Chongqing-1 and Chongqing-2 populations. Chongqing-1 was the only population showing a genetic bottleneck. Isolation by distance among colonies in the Huanggang population indicated limited alate dispersal or colony budding. Lack of isolation by distance among colonies within the populations of Changsha, Chongqing-1 and Chongqing-2, suggested long-range dispersal by alates and/or human-mediated transport. Overall, extended family colonies (73.91%) were predominant in all four populations, followed by simple (20.29%), and mixed family colonies (5.80%). Most simple families were headed by inbred related reproductive pairs in the Changsha population, while most simple families in the Chongqing-1 population were headed by outbred unrelated pairs. Simple families in the Huanggang population were a mixture of colonies headed by outbred or inbred reproductive pairs. The sample size of simple families in the Chongqing-2 population was too small to yield significant results. Extended families in all four populations were headed on the average by ≤10 neotenics. Mixed families likely originated from pleometrosis. Presence of heterozygote genotypes showed that all neotenic reproductives collected in addition from five field colonies in Wuhan city were sexually produced, suggesting that these colonies did not undergo parthenogenesis. This study contributes to better understanding of the variance of genetic structure and reproductive mode in the genus Reticulitermes.     

Chloride Is essential for capacitation and for the capacitation-associated increase in tyrosine phosphorylation

After epididymal maturation, sperm capacitation, which encompasses a complex series of molecular events, endows the sperm with the ability to fertilize an egg. This process can be mimicked in vitro in defined media, the composition of which is based on the electrolyte concentration of the oviductal fluid. It is well established that capacitation requires Na(+), HCO(3)(-), Ca(2+), and a cholesterol acceptor; however, little is known about the function of Cl(-) during this important process. To determine whether Cl(-), in addition to maintaining osmolarity, actively participates in signaling pathways that regulate capacitation, Cl(-) was replaced by either methanesulfonate or gluconate two nonpermeable anions. The absence of Cl(-) did not affect sperm viability, but capacitation-associated processes such as the increase in tyrosine phosphorylation, the increase in cAMP levels, hyperactivation, the zona pellucidae-induced acrosome reaction, and most importantly, fertilization were abolished or significantly reduced. Interestingly, the addition of cyclic AMP agonists to sperm incubated in Cl(-)-free medium rescued the increase in tyrosine phosphorylation and hyperactivation suggesting that Cl(-) acts upstream of the cAMP/protein kinase A signaling pathway. To investigate Cl(-) transport, sperm incubated in complete capacitation medium were exposed to a battery of anion transport inhibitors. Among them, bumetanide and furosemide, two blockers of Na(+)/K(+)/Cl(-) cotransporters (NKCC), inhibited all capacitation-associated events, suggesting that these transporters may mediate Cl(-) movements in sperm. Consistent with these results, Western blots using anti-NKCC1 antibodies showed the presence of this cotransporter in mature sperm.     

Sequence-specific peptide aptamers,interacting with the intracellular domain of the epidermal growth factor receptor,interfere with Stat3 activation and inhibit the growth of tumor cells

Receptor tyrosine kinases of the epidermal growth factor (EGF) receptor family regulate essential cellular functions such as proliferation, survival, migration, and differentiation but also play central roles in the etiology and progression of tumors. We have identified short peptide sequences from a random peptide library integrated into the thioredoxin scaffold protein, which specifically bind to the intracellular domain of the EGF receptor (EGFR). These molecules have the potential to selectively inhibit specific aspects of EGF receptor signaling and might become valuable as anticancer agents. Intracellular expression of the aptamer encoding gene construct KDI1 or introduction of bacterially expressed KDI1 via a protein transduction domain into EGFR-expressing cells results in KDI1.EGF receptor complex formation, a slower proliferation, and reduced soft agar colony formation. Aptamer KDI1 did not summarily block the EGF receptor tyrosine kinase activity but selectively interfered with the EGF-induced phosphorylation of the tyrosine residues 845, 1068, and 1148 as well as the phosphorylation of tyrosine 317 of p46 Shc. EGF-induced phosphorylation of Stat3 at tyrosine 705 and Stat3-dependent transactivation were also impaired. Transduction of a short synthetic peptide aptamer sequence not embedded into the scaffold protein resulted in the same impairment of EGF-induced Stat3 activation.     

Toxic HypF-N Oligomers Selectively Bind the Plasma Membrane to Impair Cell Adhesion Capability

The deposition of fibrillar protein aggregates in human organs is the hallmark of several pathological states, including highly debilitating neurodegenerative disorders and systemic amyloidoses. It is widely accepted that small oligomers arising as intermediates in the aggregation process, released by fibrils, or growing in secondary nucleation steps are the cytotoxic entities in protein-misfolding diseases, notably neurodegenerative conditions. Increasing evidence indicates that cytotoxicity is triggered by the interaction between nanosized protein aggregates and cell membranes, even though little information on the molecular details of such interaction is presently available. In this work, we propose what is, to our knowledge, a new approach, based on the use of single-cell force spectroscopy applied to multifunctional substrates, to study the interaction between protein oligomers, cell membranes, and/or the extracellular matrix. We compared the interaction of single Chinese hamster ovary cells with two types of oligomers (toxic and nontoxic) grown from the N-terminal domain of the Escherichia coli protein HypF. We were able to quantify the affinity between both oligomer type and the cell membrane by measuring the mechanical work needed to detach the cells from the aggregates, and we could discriminate the contributions of the membrane lipid and protein fractions to such affinity. The fundamental role of the ganglioside GM1 in the membrane-oligomers interaction was also highlighted. Finally, we observed that the binding of toxic oligomers to the cell membrane significantly affects the functionality of adhesion molecules such as Arg-Gly-Asp binding integrins, and that this effect requires the presence of the negatively charged sialic acid moiety of GM1.     

Inactivation of the polycomb group protein Ring1B unveils an antiproliferative role in hematopoietic cell expansion and cooperation with tumorigenesis associated with Ink4a deletion

Polycomb group (PcG) proteins act as positive regulators of cell proliferation. Ring1B is a PcG gene essential for embryonic development, but its contribution to cell turnover in regenerating tissues in not known. Here, we have generated a conditional mouse mutant line to study the Ring1B role in adult hematopoiesis. Mutant mice developed a hypocellular bone marrow that paradoxically contained an enlarged, hyperproliferating compartment of immature cells, with an intact differentiation potential. These alterations were associated with differential upregulation of cyclin D2, which occurred in all mutant bone marrow cells, and of p16^Ink4a, observed only in the differentiated compartment. Concurrent inactivation of Ink4a rescued the defective proliferation of maturing cells but did not affect the hyperproliferative activity of progenitors and resulted in a shortening of the onset of lymphomas induced by Ink4a inactivation. These data show that Ring1B restricts the progenitors' proliferation and promotes the proliferation of their maturing progeny by selectively altering the expression pattern of cell cycle regulators along hematopoietic differentiation. The novel antiproliferative role of Ring1B's downregulation of a cell cycle activator may play an important role in the tight control of hematopoietic cell turnover.