Proteasome assembly triggers a switch required for active-site maturation

The processing of propeptides and the maturation of 20S proteasomes require the association of beta rings from two half proteasomes. We propose an assembly-dependent activation model in which interactions between helix (H3 and H4) residues of the opposing half proteasomes are prerequisite for appropriate positioning of the S2-S3 loop; such positioning enables correct coordination of the active-site residue needed for propeptide cleavage. Mutations of H3 or H4 residues that participate in the association of two half proteasomes inhibit activation and prevent, in nearly all cases, the formation of full proteasomes. In contrast, mutations affecting interactions with residues of the S2-S3 loop allow the assembly of full, but activity impacted, proteasomes. The crystal structure of the inactive H3 mutant, Phe145Ala, shows that the S2-S3 loop is displaced from the position observed in wild-type proteasomes. These data support the proposed assembly-dependent activation model in which the S2-S3 loop acts as an activation switch.     

Information about transmission opportunities triggers a life-history switch in a parasite

Many microbial pathogens can switch to new hosts or adopt alternative transmission routes as environmental conditions change, displaying unexpected flexibility in their infection pathways and often causing emerging diseases. In contrast, parasitic worms that must develop through a fixed series of host species appear less likely to show phenotypic plasticity in their transmission pathways. Here, I demonstrate experimentally that a trematode parasite, Coitocaecum parvum, can accelerate its development and rapidly reach precocious maturity in its crustacean intermediate host in the absence of chemical cues emanating from its fish definitive host. Juvenile trematodes can also mature precociously when the mortality rate of their intermediate hosts is increased. Eggs produced by precocious adults hatch into viable larvae, capable of pursuing the parasite's life cycle. In the absence of chemical cues from fish hosts, the size of eggs released by precocious trematodes in their intermediate hosts becomes more variable, possibly indicating a bet-hedging strategy. These results illustrate that parasitic worms with complex life cycles have development and transmission strategies that are more plastic than commonly believed, allowing them to skip one host in their cycle when they perceive limited opportunities for transmission.     

Flipping a genetic switch by subunit exchange

Correction to: The EMBO Journal (2001) 20, 7149–7159. doi:10.1093/emboj/20.24.7149Two different structures of the anti-σ factor T4 AsiA in its dimeric state have been reported, one by us and a second by Urbauer et al (2002). The principal distinction between these structures was in the monomer fold, which displayed an approximate mirror image relationship to one another. This difference prompted a re-examination of our AsiA structure in solution, leading us to conclude that our structure of the T4 AsiA dimer was incorrectly determined in the original report. The resolution of this discrepancy was driven by the solution of the AsiA structure in two new states: a free monomer and a monomer bound to conserved region 4 from Escherichia coli σ⁷⁰ (EcSR4) (see report by Lambert et al in this issue (The EMBO Journal (2004) 23, 2952–2962). In each of these states, the chemical shifts for all atoms were similar to each other and very different from those observed in AsiA dimer, enabling a completely independent effort at the solution of the AsiA structure. The fold of AsiA determined in each of these new states was found to be very similar to that reported by Urbauer et al. Most convincing in this analysis were the 220 intermolecular NOEs observed between AsiA and EcSR4, which were only consistent with the fold reported by Urbauer et al. Both monomer states were further refined by residual dipolar coupling (RDC) analysis. We subsequently reanalyzed our original AsiA dimer NMR spectra and collected RDCs on the original dimer. No errors in chemical shift assignment were found and less than 4% of all NOEs needed to be reassigned for the fold of AsiA dimer to conform to the fold reported by Urbauer et al. This represented less than 3% of those NOEs between nonsequential residues in the polypeptide chain (17 NOEs in all); these NOEs were not previously appreciated to be ambiguous with respect to a mirror image fold. Figure 1 displays the revised structure and compares it to that of Urbauer et al. The coordinates of our structure with PDB accession code 1KA3 have been replaced. The PDB ID for the corrected AsiA dimer is 1TKV.Open in a separate windowFigure 1Comparison of recalculated structure of AsiA dimer to the structure of Urbauer et al (PDB accession code 1JR5). On the left is the recalculated structure family with a ribbon representation of the monomer shown at the bottom. On the right is the C_α trace of one model from 1JR5 and a ribbon representation at the bottom. The two structures are very similar to one another, with the exception of the position of helix A6. In 1JR5, the C-terminus is tucked against the back face of helices A1 and A4, while in our revised structures these restraints are not observed. Helix A6 in 1JR5 is also one turn shorter than that observed by us. Despite these differences, the two structures are in overall agreement.     

Auxin triggers transient local signaling for cell specification in Arabidopsis embryogenesis

The Arabidopsis embryonic root meristem is initiated by the specification of a single cell, the hypophysis. This event critically requires the antagonistic auxin response regulators MONOPTEROS and BODENLOS, but their mechanism of action is unknown. We show that these proteins interact and transiently act in a small subdomain of the proembryo adjacent to the future hypophysis. Here they promote transport of auxin, which then elicits a second response in the hypophysis itself. Our results suggest that hypophysis specification is not the direct result of a primary auxin response but rather depends on cell-to-cell signaling triggered by auxin in adjacent cells.     

Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis

During carcinogenesis of pancreatic islets in transgenic mice, an angiogenic switch activates the quiescent vasculature. Paradoxically, vascular endothelial growth factor (VEGF) and its receptors are expressed constitutively. Nevertheless, a synthetic inhibitor (SU5416) of VEGF signalling impairs angiogenic switching and tumour growth. Two metalloproteinases, MMP-2/gelatinase-A and MMP-9/gelatinase-B, are upregulated in angiogenic lesions. MMP-9 can render normal islets angiogenic, releasing VEGF. MMP inhibitors reduce angiogenic switching, and tumour number and growth, as does genetic ablation of MMP-9. Absence of MMP-2 does not impair induction of angiogenesis, but retards tumour growth, whereas lack of urokinase has no effect. Our results show that MMP-9 is a component of the angiogenic switch.     

Modeling the evolution of a classic genetic switch

Background  

The regulatory network underlying the yeast galactose-use pathway has emerged as a model system for the study of regulatory network evolution. Evidence has recently been provided for adaptive evolution in this network following a whole genome duplication event. An ancestral gene encoding a bi-functional galactokinase and co-inducer protein molecule has become subfunctionalized as paralogous genes (GAL1 and GAL3) in Saccharomyces cerevisiae, with most fitness gains being attributable to changes in cis-regulatory elements. However, the quantitative functional implications of the evolutionary changes in this regulatory network remain unexplored.     

Auxin: its role in genetic tumor induction

Seedlings of the tumor-prone amphiploid Nicotiana suaveolens X N. langsdorffii were grown on nutrient medium supplemented with indoleacetic acid (IAA) and scored at regular intervals for the incidence of tumor formation. IAA at 2 x 10(-5)m caused a significant reduction in the rate of tumor formation. Plants were also grown on nutrient medium under two different intensities of illumination, and the endogenous level of IAA was determined in 23-day-old seedlings. Those grown under 2000 ft-c of illumination had a higher incidence of tumors and a significantly lower level of endogenous IAA than those grown under 500 ft-c of illumination. A correlation in time between decline in the endogenous level of IAA and onset of tumor formation was demonstrated in greenhouse-grown plants.     

Dual selection of a genetic switch by a single selection marker

Forward engineering of synthetic genetic circuits in living cells is expected to deliver various applications in biotechnology and medicine and to provide valuable insights into the design principles of natural gene networks. However, lack of biochemical data and complexity of biological environment complicate rational design of such circuits based on quantitative simulation. Previously, we have shown that directed evolution can complement our weakness in designing genetic circuits by screening or selecting functional circuits from a large pool of nonfunctional ones. Here we describe a dual selection strategy that allows selection of both ON and OFF states of genetic circuits using tetA as a single selection marker. We also describe a successful demonstration of a genetic switch selection from a 2000-fold excess background of nonfunctional switches in three rounds of iterative selection. The dual selection system is more robust than the previously reported selection system employing three genes, with no observed false positive mutants during the simulated selections.     

Predicting the asymmetric response of a genetic switch to noise

We present a simple analytical tool which gives an approximate insight into the stationary behavior of nonlinear systems undergoing the influence of a weak and rapid noise from one dominating source, e.g. the kinetic equations describing a genetic switch with the concentration of one substrate fluctuating around a constant mean. The proposed method allows for predicting the asymmetric response of the genetic switch to noise, arising from the noise-induced shift of stationary states. The method has been tested on an example model of the lac operon regulatory network: a reduced Yildirim-Mackey model with fluctuating extracellular lactose concentration. We calculate analytically the shift of the system's stationary states in the presence of noise. The results of the analytical calculation are in excellent agreement with the results of numerical simulation of the noisy system. The simulation results suggest that the structure of the kinetics of the underlying biochemical reactions protects the bistability of the lactose utilization mechanism from environmental fluctuations. We show that, in the consequence of the noise-induced shift of stationary states, the presence of fluctuations stabilizes the behavior of the system in a selective way: Although the extrinsic noise facilitates, to some extent, switching off the lactose metabolism, the same noise prevents it from switching on.     

recA-dependent genetic switch generated by transposon Tn10

We describe a new type of regulatory switch generated in bacteriophage lambda by transposon Tn10. By this switch, phage genes alternate reversibly between expressed and non-expressed states as the direct consequence of a reversible DNA rearrangement. The switch itself has arisen via Tn10-promoted recombination. The subsequent “flip-flop” in gene expression occurs by general recombination between two IS10 elements serving as “portable regions of homology”.     

Casein Kinase II phosphorylation-induced conformational switch triggers degradation of the papillomavirus E2 protein

The major phosphorylation sites of the bovine papillomavirus E2 transactivator protein are two serine residues, 298 and 301, that are located in a flexible hinge region between the DNA binding and transactivation domains. Phosphorylation of serine residue 301 promotes ubiquitination and rapid degradation of the E2 protein by the proteasome pathway. To understand the mechanism through which phosphorylation regulates the intracellular levels of this unique papillomavirus regulatory protein, we have carried out an extensive mutational analysis of the region surrounding the phosphorylation sites of the E2 protein. Our results indicate that casein kinase II phosphorylates serine 301. However, phosphorylation of serine 301 is not a sufficient recognition motif for proteasomal degradation; other residues that directly surround the phosphorylation sites are crucial for E2 degradation. The phenotypes of E2 proteins mutated in this region indicate that phosphorylation of serine 301 induces a conformational change that leads to degradation of the E2 protein. In support of this model, circular dichroism studies of the conformational tendencies of peptides from this region indicate that phosphorylation at position 301 decreases the local thermodynamic stability of this region. Thus, this region appears to have evolved to display a marginal local thermodynamic stability that can be regulated by phosphorylation, leading to targeted degradation of the E2 protein.     

A genetic bistable switch utilizing nonlinear protein degradation

Background

Fluorescent reporter proteins have revolutionized our understanding of cellular bioprocesses by enabling live cell imaging with exquisite spatio-temporal resolution. Existing fluorescent proteins are predominantly based on the green fluorescent protein (GFP) and related analogs. However, GFP-family proteins strictly require molecular oxygen for maturation of fluorescence, which precludes their application for investigating biological processes in low-oxygen environments. A new class of oxygen-independent fluorescent reporter proteins was recently reported based on flavin-binding photosensors from Bacillus subtilis and Pseudomonas putida. However, flavin-binding fluorescent proteins show very limited brightness, which restricts their utility as biological imaging probes.

Results

In this work, we report the discovery of bright mutants of a flavin-binding fluorescent protein from P. putida using directed evolution by site saturation mutagenesis. We discovered two mutations at a chromophore-proximal amino acid (F37S and F37T) that confer a twofold enhancement in brightness relative to the wild type fluorescent protein through improvements in quantum yield and holoprotein fraction. In addition, we observed that substitution with other aromatic amino acids at this residue (F37Y and F37W) severely diminishes fluorescence emission. Therefore, we identify F37 as a key amino acid residue in determining fluorescence.

Conclusions

To increase the scope and utility of flavin-binding fluorescent proteins as practical fluorescent reporters, there is a strong need for improved variants of the wild type protein. Our work reports on the application of site saturation mutagenesis to isolate brighter variants of a flavin-binding fluorescent protein, which is a first-of-its-kind approach. Overall, we anticipate that the improved variants will find pervasive use as fluorescent reporters for biological studies in low-oxygen environments.