Optimization of the cellular import of functionally active SH2-domain-interacting phosphopeptides

Phosphopeptides interacting with src homology 2 (SH2) domains can activate essential signaling enzymes in vitro. When delivered to cells, they may disrupt protein-protein interactions, thereby influencing intracellular signaling. We showed earlier that phosphopeptides corresponding to the inhibitory motif of Fcγ receptor IIb and a motif of the Grb2-associated binder 1 adaptor protein activate SH2-containing tyrosine phosphatase 2 in vitro. To study the ex vivo effects of these peptides, we have now compared different methods for peptide delivery: (i) permeabilization of the target cells and (ii) the use of cell-permeable vectors, which are potentially able to transport biologically active compounds into B cells. We found octanoyl-Arg₈ to be an optimal carrier for the delivery of phosphopeptides to the cells. With this strategy, the function of cell-permeable SHP-2-binding phosphopeptides was analyzed. These peptides modulated the protein phosphorylation in B cells in a dose- and time-dependent manner. Received 27 July 2006; received after revision 4 September 2006; accepted 18 September 2006     

Atomic structure holography using thermal neutrons.

The idea of atomic-resolution holography has its roots in the X-ray work of Bragg and in Gabor's electron interference microscope. Gabor's lensless microscope was not realized in his time, but over the past twelve years there has been a steady increase in the number of reports on atomic-resolution holography. All of this work involves the use of electrons or hard X-rays to produce the hologram. Neutrons are often unique among scattering probes in their interaction with materials: for example, the relative visibility of hydrogen and its isotopes is a great advantage in the study of polymers and biologically relevant materials. Recent work proposed that atomic-resolution holography could be achieved with thermal neutrons. Here we use monochromatic thermal neutrons, adopting the inside-source concept of Sz?ke, to image planes of oxygen atoms located above and below a single hydrogen atom in the oxide mineral simpsonite.     

Maternal control of resting-egg production in Daphnia.

Many planktonic organisms produce 'resting' stages when the environmental conditions deteriorate. Like seeds, resting stages can survive unfavourable conditions. The crustacean Daphnia normally reproduces by means of parthenogenetically produced normal, not resting, eggs-but occasionally switches to bisexual reproduction, which results in two resting eggs encased in a robust structure carried on the back of the female. This 'ephippium' is shed with the next moult, and can survive dormant for many years. The induction of resting-egg production requires multiple environmental stimuli, one of them being photoperiod. The switch from production of parthenogenetic eggs to resting eggs in Daphnia has recently been shown to be influenced by a maternal food effect. Here we present evidence that female Daphnia transmit information not only about food but also on photoperiod to their offspring, and influence the production of resting eggs in the next generation. The combined maternal effects can be relevant for the correct timing of resting-egg production-for example, in discriminating between spring and autumn conditions.     

Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB-MoaD complex.

The activation of ubiquitin and related protein modifiers is catalysed by members of the E1 enzyme family that use ATP for the covalent self-attachment of the modifiers to a conserved cysteine. The Escherichia coli proteins MoeB and MoaD are involved in molybdenum cofactor (Moco) biosynthesis, an evolutionarily conserved pathway. The MoeB- and E1-catalysed reactions are mechanistically similar, and despite a lack of sequence similarity, MoaD and ubiquitin display the same fold including a conserved carboxy-terminal Gly-Gly motif. Similar to the E1 enzymes, MoeB activates the C terminus of MoaD to form an acyl-adenylate. Subsequently, a sulphurtransferase converts the MoaD acyl-adenylate to a thiocarboxylate that acts as the sulphur donor during Moco biosynthesis. These findings suggest that ubiquitin and E1 are derived from two ancestral genes closely related to moaD and moeB. Here we present the crystal structures of the MoeB-MoaD complex in its apo, ATP-bound, and MoaD-adenylate forms, and highlight the functional similarities between the MoeB- and E1-substrate complexes. These structures provide a molecular framework for understanding the activation of ubiquitin, Rub, SUMO and the sulphur incorporation step during Moco and thiamine biosynthesis.     

Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms.

The stable propagation of genetic information requires that the entire genome of an organism be faithfully replicated once and only once each cell cycle. In eukaryotes, this replication is initiated at hundreds to thousands of replication origins distributed over the genome, each of which must be prohibited from re-initiating DNA replication within every cell cycle. How cells prevent re-initiation has been a long-standing question in cell biology. In several eukaryotes, cyclin-dependent kinases (CDKs) have been implicated in promoting the block to re-initiation, but exactly how they perform this function is unclear. Here we show that B-type CDKs in Saccharomyces cerevisiae prevent re-initiation through multiple overlapping mechanisms, including phosphorylation of the origin recognition complex (ORC), downregulation of Cdc6 activity, and nuclear exclusion of the Mcm2-7 complex. Only when all three inhibitory pathways are disrupted do origins re-initiate DNA replication in G2/M cells. These studies show that each of these three independent mechanisms of regulation is functionally important.     

Domains specifying thrombin-receptor interaction.

Platelet activation by the coagulation protease thrombin is central to arterial thrombosis, a major cause of morbidity and mortality. We recently isolated a complementary DNA encoding the platelet thrombin receptor. The extracellular amino-terminal extension of this seven transmembrane domain receptor contains the putative thrombin cleavage site LDPR/S which is critical for receptor activation. By replacing this cleavage site with the cleavage site for enterokinase, we have created a functional enterokinase receptor. This result demonstrates that all information necessary for receptor activation is provided by receptor proteolysis. Nanomolar enterokinase concentrations are required to activate this new receptor, in contrast to the picomolar thrombin concentrations that activate wild-type thrombin receptor. We identified a receptor domain critical for thrombin's remarkable potency at its receptor. This domain resembles the carboxyl tail of the leech anticoagulant hirudin and functions by binding to thrombin's anion-binding exosite. Our studies thus define a model for thrombin-receptor interaction. The utility of this model was demonstrated by the design of novel thrombin inhibitors based on receptor peptides.     

High-affinity NGF binding requires coexpression of the trk proto-oncogene and the low-affinity NGF receptor.

Nerve growth factor (NGF) interacts with two different low-affinity receptors that can be distinguished by affinity crosslinking. Reconstitution experiments by membrane fusion and transient transfection into heterologous cells indicate that high-affinity NGF binding requires coexpression and binding to both the low-affinity NGF receptor and the tyrosine kinase trk gene product. These studies reveal a new growth factor receptor-mediated mechanism of cellular differentiation involving trk and the low-affinity NGF receptor.     

Running energetics in the pronghorn antelope

The pronghorn antelope (Antilocapra americana) has an alleged top speed of 100 km h-1, second only to the cheetah (Acionyx jubatus) among land vertebrates, a possible response to predation in the exposed habitat of the North American prairie. Unlike cheetahs, however, pronghorn antelope are distance runners rather than sprinters, and can run 11 km in 10 min, an average speed of 65 km h-1. We measured maximum oxygen uptake in pronghorn antelope to distinguish between two potential explanations for this ability: either they have evolved a uniquely high muscular efficiency (low cost of transport) or they can supply oxygen to the muscles at unusually high levels. Because the cost of transport (energy per unit distance covered per unit body mass) varies as a predictable function of body mass among terrestrial vertebrates, we can calculate the predicted cost to maintain speeds of 65 and 100 km h-1 in an average 32-kg animal. The resulting range of predicted values, 3.2-5.1 ml O2 kg-1 s-1, far surpasses the predicted maximum aerobic capacity of a 32-kg mammal (1.5 ml O2 kg-1 s-1). We conclude that their performance is achieved by an extraordinary capacity to consume and process enough oxygen to support a predicted running speed greater than 20 ms-1 (70 km h-1), attained without unique respiratory-system structures.