Evidence for regulation of protein synthesis at the elongation step by CDK1/cyclin B phosphorylation

Eukaryotic elongation factor 1 (eEF-1) contains the guanine nucleotide exchange factor eEF-1B that loads the G protein eEF-1A with GTP after each cycle of elongation during protein synthesis. Two features of eEF-1B have not yet been elucidated: (i) the presence of the unique valyl-tRNA synthetase; (ii) the significance of target sites for the cell cycle protein kinase CDK1/cyclin B. The roles of these two features were addressed by elongation measurements in vitro using cell-free extracts. A poly(GUA) template RNA was generated to support both poly(valine) and poly(serine) synthesis and poly(phenylalanine) synthesis was driven by a poly(uridylic acid) template. Elongation rates were in the order phenylalanine > valine > serine. Addition of CDK1/cyclin B decreased the elongation rate for valine whereas the rate for serine and phenylalanine elongation was increased. This effect was correlated with phosphorylation of the eEF-1delta and eEF-1gamma subunits of eEF-1B. Our results demonstrate specific regulation of elongation by CDK1/cyclin B phosphorylation.     

Divergent N-terminal sequences target an inducible testis deubiquitinating enzyme to distinct subcellular structures

Ubiquitin-specific processing proteases (UBPs) presently form the largest enzyme family in the ubiquitin system, characterized by a core region containing conserved motifs surrounded by divergent sequences, most commonly at the N-terminal end. The functions of these divergent sequences remain unclear. We identified two isoforms of a novel testis-specific UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini, thereby permitting dissection of the functions of these two regions. Both isoforms were germ cell specific and developmentally regulated. Immunocytochemistry revealed that UBP-t1 was induced in step 16 to 19 spermatids while UBP-t2 was expressed in step 18 to 19 spermatids. Immunoelectron microscopy showed that UBP-t1 was found in the nucleus while UBP-t2 was extranuclear and was found in residual bodies. For the first time, we show that the differential subcellular localization was due to the distinct N-terminal sequences. When transfected into COS-7 cells, the core region was expressed throughout the cell but the UBP-t1 and UBP-t2 isoforms were concentrated in the nucleus and the perinuclear region, respectively. Fusions of each N-terminal end with green fluorescent protein yielded the same subcellular localization as the native proteins, indicating that the N-terminal ends were sufficient for determining differential localization. Interestingly, UBP-t2 colocalized with anti-gamma-tubulin immunoreactivity, indicating that like several other components of the ubiquitin system, a deubiquitinating enzyme is associated with the centrosome. Regulated expression and alternative N termini can confer specificity of UBP function by restricting its temporal and spatial loci of action.     

Proteolysis of the exodomain of recombinant protease-activated receptors: prediction of receptor activation or inactivation by MALDI mass spectrometry

Protease-activated receptors (PARs) mediate cell activation after proteolytic cleavage of their extracellular amino terminus. Thrombin selectively cleaves PAR1, PAR3, and PAR4 to induce activation of platelets and vascular cells, while PAR2 is preferentially cleaved by trypsin. In pathological situations, other proteolytic enzymes may be generated in the circulation and could modify the responses of PARs by cleaving their extracellular domains. To assess the ability of such proteases to activate or inactivate PARs, we designed a strategy for locating cleavage sites on the exofacial NH(2)-terminal fragments of the receptors. The first extracellular segments of PAR1 (PAR1E) and PAR2 (PAR2E) expressed as recombinant proteins in Escherichia coli were incubated with a series of proteases likely to be encountered in the circulation during thrombosis or inflammation. Kinetic and dose-response studies were performed, and the cleavage products were analyzed by MALDI-TOF mass spectrometry. Thrombin cleaved PAR1E at the Arg41-Ser42 activation site at concentrations known to induce cellular activation, supporting a native conformation of the recombinant polypeptide. Plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3 cleaved at multiple sites and would be expected to disable PAR1 by cleaving COOH-terminal to the activation site. Cleavage specificities were further confirmed using activation site defective PAR1E S42P mutant polypeptides. Surface plasmon resonance studies on immobilized PAR1E or PAR1E S42P were consistent with cleavage results obtained in solution and allowed us to determine affinities of PAR1E-thrombin binding. FACS analyses of intact platelets confirmed the cleavage of PAR1 downstream of the Arg41-Ser42 site. Mass spectrometry studies of PAR2E predicted activation of PAR2 by trypsin through cleavage at the Arg36-Ser37 site, no effect of thrombin, and inactivation of the receptor by plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3. The inhibitory effect of elastase was confirmed on native PAR1 and PAR2 on the basis of Ca(2+) signaling studies in endothelial cells. It was concluded that none of the main proteases generated during fibrinolysis or inflammation appears to be able to signal through PAR1 or PAR2. This strategy provides results which can be extended to the native receptor to predict its activation or inactivation, and it could likewise be used to study other PARs or protease-dependent processes.     

Bibliography

Bibliography

Bibliography     

Capsaicin-treated rats permanently overingest low- but not high-concentration sucrose solutions

The effect of capsaicin-induced chemical ablation of visceral afferents on 1-h liquid sucrose consumption was investigated in food-deprived rats. We first show that although 10% sucrose is permanently overconsumed by capsaicin-treated (CAPs) compared with vehicle-treated (VEHs) control rats, 40% sucrose is only overconsumed during the first but not subsequent 1-h exposures. Furthermore, one group of CAPs lost the overconsumption response at 20% when exposed to progressively increasing sucrose concentrations of 10-40%, and another group recovered the overconsumption response at 10% when exposed to a series of decreasing concentrations. Control rats ingested relatively constant volumes of sucrose over the range of 10, 15, and 20%, resulting in significantly different energy intakes. In contrast, CAPs generally showed a concentration-dependent decrease in volume intake, resulting in relatively constant energy intake. These results suggest that capsaicin-sensitive visceral afferents, likely from gastric distension and other preabsorptive sensors, provide major control over volume ingested. In the absence of these signals, rats initially overconsume, but rapidly learn to use other signals from capsaicin-resistant preabsorptive or postabsorptive sites, to control future intake. This redundant satiety system appears to be sensitive to the osmotic value or caloric content of the unfamiliar food, but only if this is above a threshold of about 15% sucrose.     

Estimating phylogenetic inertia in Tithonia (Asteraceae): a comparative approach

Abstract.— Phylogenetic inertia is a difficult issue in evolutionary biology because we have yet to reach a consensus about how to measure it. In this study a comparative approach is used to evaluate phylogenetic inertia in 14 demographic and morphological characters in 10 species and one subspecies of the genus Tithonia (Asteraceae). Three different methods, autocorrelational analysis, phylogenetic correlograms, and ancestor-state reconstruction, were used to evaluate phylogenetic inertia in these traits. Results were highly dependent on the method applied. Autoregression and phylogenetic eigenvector regression (PVR) methods found more inertia in morphological traits. In contrast, phylogenetic correlograms and ancestor-state reconstruction suggest that morphological characters exhibit less phylogenetic inertia than demographic ones. The differences between results are discussed and methods are compared in an effort to understand phylogenetic inertia more thoroughly.