Closely related G-protein-coupled receptors use multiple and distinct domains on G-protein alpha-subunits for selective coupling

The molecular basis of selectivity in G-protein receptor coupling has been explored by comparing the abilities of G-protein heterotrimers containing chimeric Galpha subunits, comprised of various regions of Gi1alpha, Gtalpha, and Gqalpha, to stabilize the high affinity agonist binding state of serotonin, adenosine, and muscarinic receptors. The data indicate that multiple and distinct determinants of selectivity exist for individual receptors. While the A1 adenosine receptor does not distinguish between Gi1alpha and Gtalpha sequences, the 5-HT1A and 5-HT1B serotonin and M2 muscarinic receptors can couple with Gi1 but not Gt. It is possible to distinguish domains that eliminate coupling and are defined as "critical," from those that impair coupling and are defined as "important." Domains within the N terminus, alpha4-helix, and alpha4-helix-alpha4/beta6-loop of Gi1alpha are involved in 5-HT and M2 receptor interactions. Chimeric Gi1alpha/Gqalpha subunits verify the critical role of the Galpha C terminus in receptor coupling, however, the individual receptors differ in the C-terminal amino acids required for coupling. Furthermore, the EC50 for interactions with Gi1 differ among the individual receptors. These results suggest that coupling selectivity ultimately involves subtle and cooperative interactions among various domains on both the G-protein and the associated receptor as well as the G-protein concentration.     

Novel bi- and trifunctional inhibitors of tumor-associated proteolytic systems

Serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) are involved in cancer cell invasion and metastasis. Recently, a recombinant bifunctional inhibitor (chCys-uPA19-31) directed against cysteine proteases and the urokinase-type plasminogen activator (uPA)/plasmin serine protease system was generated by introducing the uPA receptor (uPAR)-binding site of uPA into chicken cystatin (chCysWT). In the present study, we designed and recombinantly produced multifunctional inhibitors also targeting MMPs. The inhibitors comprise the N-terminal inhibitory domain of human TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) or TIMP-3, fused to chCys-uPA19-31 or chCysWT. As demonstrated by various techniques, these fusion proteins effectively interfere with all three targeted protease systems. In in vitro Matrigel invasion assays, the addition of recombinant inhibitors strongly reduced invasion of ovarian cancer cells (OV-MZ-6#8). Additionally, OV-MZ-6#8 cells were stably transfected with expression plasmids encoding the various inhibitors. Synthesis and secretion of the inhibitors was verified by a newly developed ELISA, which selectively detects the recombinant proteins. Invasive capacity of inhibitor-producing cells was significantly reduced compared to vector-transfected control cells. Thus, these novel, compact, and small-size inhibitors directed against up to three different tumor-associated proteolytic systems may represent promising agents for prevention of tumor cell migration and metastasis.     

Transferable domain in the G(1) cyclin Cln2 sufficient to switch degradation of Sic1 from the E3 ubiquitin ligase SCF(Cdc4) to SCF(Grr1)

Degradation of Saccharomyces cerevisiae G(1) cyclins Cln1 and Cln2 is mediated by the ubiquitin-proteasome pathway and involves the SCF E3 ubiquitin-ligase complex containing the F-box protein Grr1 (SCF(Grr1)). Here we identify the domain of Cln2 that confers instability and describe the signals in Cln2 that result in binding to Grr1 and rapid degradation. We demonstrate that mutants of Cln2 that lack a cluster of four Cdc28 consensus phosphorylation sites are highly stabilized and fail to interact with Grr1 in vivo. Since one of the phosphorylation sites lies within the Cln2 PEST motif, a sequence rich in proline, aspartate or glutamate, serine, and threonine residues found in many unstable proteins, we fused various Cln2 C-terminal domains containing combinations of the PEST and the phosphoacceptor motifs to stable reporter proteins. We show that fusion of the Cln2 domain to a stabilized form of the cyclin-dependent kinase inhibitor Sic1 (Delta N-Sic1), a substrate of SCF(Cdc4), results in degradation in a phosphorylation-dependent manner. Fusion of Cln2 degradation domains to Delta N-Sic1 switches degradation of Sic1 from SCF(Cdc4) to SCF(Grr1). Delta N-Sic1 fused with a Cln2 domain containing the PEST motif and four phosphorylation sites binds to Grr1 and is unstable and ubiquitinated in vivo. Interestingly, the phosphoacceptor domain of Cln2 binds to Grr1 but is not ubiquitinated and is stable. In summary, we have identified a small transferable domain in Cln2 that can redirect a stabilized SCF(Cdc4) target for SCF(Grr1)-mediated degradation by the ubiquitin-proteasome pathway.     

Drosophila melanogaster FMRFamide-containing peptides: redundant or diverse functions?

FMRFamide-related peptides (FaRPs) are expressed throughout the animal kingdom and regulate a multitude of physiological activities. FaRPs have an RFamide C-terminal consensus structure that is important for interaction with the receptor. The ease of genetic manipulation and availability of genomic sequences makes Drosophila melanogaster an important experimental organism. Multiple classes of FaRPs encoded by different genes have been identified within this species. Here, we review FMRFamide-containing peptides encoded by the D. melanogaster FMRFamide gene in order to review the data on the expression, regulation, and activity of these peptides as well as acknowledge further endeavors required to elucidate FaRP signaling.     

Molecular basis for the recognition of a nonclassical nuclear localization signal by importin beta

Nuclear import of proteins containing a classical nuclear localization signal (NLS) involves NLS recognition by importin alpha, which associates with importin beta via the IBB domain. Other proteins, including parathyroid hormone-related protein (PTHrP), are imported into the nucleus by direct interaction with importin beta. We solved the crystal structure of a fragment of importin beta-1 (1-485) bound to the nonclassical NLS of PTHrP. The structure reveals a second extended cargo binding site on importin beta distinct from the IBB domain binding site. Using a permeabilized cell import assay we demonstrate that importin beta (1-485) can import PTHrP-coupled cargo in a Ran-dependent manner. We propose that this region contains a prototypical nuclear import receptor domain, which could have evolved into the modern importin beta superfamily.     

FERTILIZATION SUCCESS CAN DRIVE PATTERNS OF PHASE DOMINANCE IN COMPLEX LIFE HISTORIES1

Many algal life cycles alternate between two free‐living generations. Life histories in which the two generations look identical (isomorphic) are common, particularly in the Rhodophyta. Reports of natural populations dominated by one generation of the life history have sought explanation in terms of phase‐specific differences in mortality and reproductive output, yet in many cases identification of these adaptations has been elusive or inconsistent with predictions. We hypothesized that the gametophyte‐to‐sporophyte ratio of ecologically equivalent isomorphs could result from variation in fertilization rate. We developed two models to test this hypothesis: one representing a generalized isomorphic life history and the other specific to red algae with a Polysiphonia‐type life history. Fertilization rate affected the gametophyte‐to‐sporophyte ratio, especially at low fertilization rates. In the general model, gametophytes dominated the population regardless of fertilization rate unless egg production greatly exceeded meiospore production. In the red algal model, phase dominance depended on the combination of fertilization rate and the number of carpospores produced per fertilization. The generational composition of model multiphasic algal populations results from their inherent reproductive characteristics and the dynamic environment to which fertilization and mortality rates are tied.     

Functionally Referential Alarm Calls in Tamarins (Saguinus fuscicollis and Saguinus mystax) – Evidence from Playback Experiments

Studies on primate vocalisation have revealed different types of alarm call systems ranging from graded signals based on response urgency to functionally referential alarm calls that elicit predator‐specific reactions. In addition, alarm call systems that include both highly specific and other more unspecific calls have been reported. There has been consistent discussion on the possible factors leading to the evolution of different alarm call systems, among which is the need of qualitatively different escape strategies. We studied the alarm calls of free‐ranging saddleback and moustached tamarins (Saguinus fuscicollis and Saguinus mystax) in northeast Peru. Both species have predator‐specific alarm calls and show specific non‐vocal reactions. In response to aerial predators, they look upwards and quickly move downwards, while in response to terrestrial predators, they look downwards and sometimes approach the predator. We conducted playback experiments to test if the predator‐specific reactions could be elicited in the absence of the predator by the tamarins’ alarm calls alone. We found that in response to aerial alarm call playbacks the subjects looked significantly longer upwards, and in response to terrestrial alarm call playbacks they looked significantly longer downwards. Thus, the tamarins reacted as if external referents, i.e. information about the predator type or the appropriate reaction, were encoded in the acoustic features of the calls. In addition, we found no differences in the responses of S. fuscicollis and S. mystax whether the alarm call stimulus was produced by a conspecific or a heterospecific caller. Furthermore, it seems that S. fuscicollis terrestrial alarm calls were less specific than either S. mystax terrestrial predator alarms or either species’ aerial predator alarms, but because of the small sample size it is difficult to draw a final conclusion.     

Isolation and characterization of diazotrophic bacteria from banana and pineapple plants

Banana and pineapple fruit crops are widely cultivated in tropical areas where high amounts of fertilizers are applied, principally nitrogen. Over 200 kg N.ha^-1.yr^-1 is often applied to these crops. Nevertheless, developing countries face the problem of high costs of chemical fertilizers. As already demonstrated for other tropical crops, like sugar cane, the utilization of nitrogen-fixing bacteria may support the growth of these fruit plants. In this work, we demonstrate the association of nitrogen-fixing bacteria with banana and pineapple. Samples from roots, stems, leaves and fruits of different genotypes showed the occurrence of diazotrophic bacteria, when evaluated in semi-specific semi-solid media. These isolates could be separated into seven different groups according to their morphological and physiological characteristics. Additional, phylogenetic assignments were performed with group- and species-specific oligonucleotide probes. Bacteria related to the groups of Azospirillum amazonense, Azospirillum lipoferum, Burkholderia sp. and a group similar to the genus Herbaspirillum could be detected in samples of both crops. However, Azospirillum brasilense and another two groups of Herbaspirillum-like bacteria were detected only in banana plants. Two isolates of the latter group were identified as Herbaspirillum seropedicae, whereas the other isolates may represent a new Herbaspirillum species.     

pH-dependent conformational flexibility of the SARS-CoV main proteinase (M(pro)) dimer: molecular dynamics simulations and multiple X-ray structure analyses

The SARS coronavirus main proteinase (M(pro)) is a key enzyme in the processing of the viral polyproteins and thus an attractive target for the discovery of drugs directed against SARS. The enzyme has been shown by X-ray crystallography to undergo significant pH-dependent conformational changes. Here, we assess the conformational flexibility of the M(pro) by analysis of multiple crystal structures (including two new crystal forms) and by molecular dynamics (MD) calculations. The MD simulations take into account the different protonation states of two histidine residues in the substrate-binding site and explain the pH-activity profile of the enzyme. The low enzymatic activity of the M(pro) monomer and the need for dimerization are also discussed.