Geldanamycins trigger a novel Ron degradative pathway, hampering oncogenic signaling

Ron, the tyrosine kinase receptor for macrophage-stimulating protein is responsible for proliferation and migration of cells from different tissues. Ron can acquire oncogenic potential by single point mutations in the kinase domain, and dysregulated Ron signaling has been involved in the development of different human cancers. We have previously shown that ligand-activated Ron recruits the negative regulator c-Cbl, which mediates its ubiquitylation and degradation. Here we report that Ron is ubiquitylated also by the U-box E3 ligase C-terminal Hsc70-interacting protein (CHIP), recruited via chaperone intermediates Hsp90 and Hsc70. Gene silencing shows that CHIP activity is necessary to mediate Ron degradation upon cell treatment with Hsp90 inhibitors geldanamycins. The oncogenic Ron(M1254T) receptor escapes from c-Cbl negative regulation but retains a strong association with CHIP. This constitutively active mutant of Ron displays increased sensitivity to geldanamycins, enhanced physical interaction with Hsp90, and more rapid degradation rate. Cell growth and migration, as well as the transforming potential evoked by Ron(M1254T), are abrogated upon Hsp90 inhibition. These data highlight a novel mechanism for Ron degradation and propose Hsp90 antagonists like geldanamycins as suitable pharmacological agents for therapy of cancers where altered Ron signaling is involved.     

The signaling protein MucG negatively affects the production and the molecular mass of alginate in Azotobacter vinelandii

Azotobacter vinelandii is a soil bacterium that produces the polysaccharide alginate. In this work, we identified a miniTn5 mutant, named GG9, which showed increased alginate production of higher molecular mass, and increased expression of the alginate biosynthetic genes algD and alg8 when compared to its parental strain. The miniTn5 was inserted within ORF Avin07920 encoding a hypothetical protein. Avin07910, located immediately downstream and predicted to form an operon with Avin07920, encodes an inner membrane multi-domain signaling protein here named mucG. Insertional inactivation of mucG resulted in a phenotype of increased alginate production of higher molecular mass similar to that of mutant GG9. The MucG protein contains a periplasmic and putative HAMP and PAS domains, which are linked to GGDEF and EAL domains. The last two domains are potentially involved in the synthesis and degradation, respectively, of bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP), a secondary messenger that has been reported to be essential for alginate production. Therefore, we hypothesized that the negative effect of MucG on the production of this polymer could be explained by the putative phosphodiesterase activity of the EAL domain. Indeed, we found that alanine replacement mutagenesis of the MucG EAL motif or deletion of the entire EAL domain resulted in increased alginate production of higher molecular mass similar to the GG9 and mucG mutants. To our knowledge, this is the first reported protein that simultaneous affects the production of alginate and its molecular mass.

     

Contribution of inter-subunit interactions to the thermostability of <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis> citrate synthase

Using citrate synthase from the hyperthermophile Pyrococcus furiosus (PfCS) as our test molecule, we show through guanidine hydrochloride-induced unfolding that the dimer separates into folded, but inactive, monomers before individual subunit unfolding takes place. Given that forces across the dimer interface are vital for thermostability, a robust computational method was derived that uses the University of Houston Brownian Dynamics (UHBD) program to calculate both the hydrophobic and electrostatic contribution to the dimerisation energy at 100°C. The results from computational and experimental determination of the lowered stability of interface mutants were correlated, being both of the same order of magnitude and placing the mutant proteins in the same order of stability. This computational method, optimised for hyperthermophilic molecules and tested in the laboratory, after further testing on other examples, could be of widespread use in the prediction of thermostabilising mutations in other oligomeric proteins for which dissociation is the first step in unfolding.     

Genetic differentiation that is exceptionally high and unexpectedly sensitive to geographic distance in the absence of gene flow: Insights from the genus Eranthis in East Asian regions

Genetic differentiation between populations is determined by various factors, including gene flow, selection, mutation, and genetic drift. Among these, gene flow is known to counter genetic differentiation. The genus Eranthis, an early flowering perennial herb, can serve as a good model to study genetic differentiation and gene flow due to its easily detectable population characteristics and known reproductive strategies, which can be associated with gene flow patterns. Eranthis populations are typically small and geographically separated from the others. Moreover, previous studies and our own observations suggest that seed and pollen dispersal between Eranthis populations is highly unlikely and therefore, currently, gene flow may not be probable in this genus. Based on these premises, we hypothesized that the genetic differentiation between the Eranthis populations would be significant, and that the genetic differentiation would not sensitively reflect geographic distance in the absence of gene flow. To test these hypotheses, genetic differentiation, genetic distance, isolation by distance, historical gene flow, and bottlenecks were analyzed in four species of this genus. Genetic differentiation was significantly high, and in many cases, extremely high. Moreover, genetic differentiation and geographic distance were positively correlated in most cases. We provide possible explanations for these observations. First, we suggest that the combination of the marker type used in our study (chloroplast microsatellites), genetic drift, and possibly selection might have resulted in the extremely high genetic differentiation observed herein. Additionally, we provide the possibility that genetic distance reflects geographic distance through historical gene flow, or adaptation in the absence of historical gene flow. Nevertheless, our explanations can be more rigorously examined and further refined through additional observations and various population genetic analyses. In particular, we suggest that other accessible populations of the genus Eranthis should be included in future studies to better characterize the intriguing population dynamics of this genus.     

Formation of the hydrophobic core of ribonuclease A through sequential coordinated conformational transitions

With steady-state and time-resolved fluorescence energy-transfer measurements, we determined the distributions of intramolecular distances in nine mutants to study the conformations of wild-type ribonuclease A in the reduced state under folding conditions. Although far-UV-CD measurements show no evidence for a secondary-structure transition, temperature- and GdnHCl-induced changes in intramolecular distance distributions in the reduced state revealed evidence for long-range subdomain structures in the denatured protein. These poorly defined structures, reflected here by wide distributions corresponding to a wide range of energies, form during refolding in a complex sequence of multiple subdomain transitions. A more well-defined structure emerges only when this structural framework, which directs the successive steps in the folding process, matures and is reinforced by stronger interactions such as disulfide bonds.