A hybrid pathway for self-sustained luminescence.

The fungal bioluminescence pathway can be reconstituted in other organisms allowing luminescence imaging without exogenously supplied substrate. The pathway starts from hispidin biosynthesis-a step catalyzed by a large fungal polyketide synthase that requires a posttranslational modification for activity. Here, we report identification of alternative compact hispidin synthases encoded by a phylogenetically diverse group of plants. A hybrid bioluminescence pathway that combines plant and fungal genes is more compact, not dependent on availability of machinery for posttranslational modifications, and confers autonomous bioluminescence in yeast, mammalian, and plant hosts. The compact size of plant hispidin synthases enables additional modes of delivery of autoluminescence, such as delivery with viral vectors.

An improved pathway for autonomous bioluminescence imaging in eukaryotes.

The discovery of the bioluminescence pathway in the fungus Neonothopanus nambi enabled engineering of eukaryotes with self-sustained luminescence. However, the brightness of luminescence in heterologous hosts was limited by performance of the native fungal enzymes. Here we report optimized versions of the pathway that enhance bioluminescence by one to two orders of magnitude in plant, fungal and mammalian hosts, and enable longitudinal video-rate imaging.

Systematic Comparison of Plant Promoters in Nicotiana spp. Expression Systems.

We report a systematic comparison of 19 plant promoters and 20 promoter-terminator combinations in two expression systems: agroinfiltration in Nicotiana benthamiana leaves, and Nicotiana tabacum BY-2 plant cell packs. The set of promoters tested comprised those not present in previously published work, including several computationally predicted synthetic promoters validated here for the first time. The expression of EGFP driven by different promoters varied by more than two orders of magnitude and was largely consistent between two tested Nicotiana systems. We confirmed previous reports of significant modulation of expression by terminators, as well as synergistic effects of promoters and terminators. Additionally, we observed non-linear effects of gene dosage on expression level. The dataset presented here can inform the design of genetic constructs for plant engineering and transient expression assays.

Carbonic anhydrase amyloid fibrils composed of laterally associated protofilaments show reduced cytotoxicity.

Cytotoxicity of amyloid fibrils has been shown to depend on their structure. However, specific features of toxic and non-toxic amyloids remain unclear. Here we focus on the relationship between structural characteristics of the fibrils and their cytotoxicity. Bovine carbonic anhydrase B (BCAB) serves as the object of this study because its amyloids reduce cell viability. Limited proteolysis and mass spectrometry were used to determine BCAB regions forming the core of amyloid fibrils. Four BCAB mutants with substitutions reducing hydrophobicity in the regions important for amyloid formation were obtained to study the kinetics of aggregation, structural features, and cytotoxicity of the amyloids. We demonstrate that fibrils of WT BCAB, L78A, L139A, and M239A variants display a pronounced toxic effect on eukaryotic cells, while I208A mutation significantly reduces the cell-damaging effect of amyloids. The data obtained conclude that cytotoxicity of BCAB fibrils does not depend on their length, secondary structure, and exposure of hydrophobic groups to the solvent. A distinctive feature of the low-toxic I208A fibrils is their specific morphology characterized by the lateral protofilaments association and formation of fibril-ribbons.

Annotation of the local context of the RNA secondary structure improves the classification and prediction of A-minors.

Non-coding RNAs play a crucial role in various cellular processes in living organisms, and RNA functions heavily depend on molecule structures composed of stems, loops, and various tertiary motifs. Among those, the most frequent are A-minor interactions, which are often involved in the formation of more complex motifs such as kink-turns and pseudoknots. We present a novel classification of A-minors in terms of RNA secondary structure where each nucleotide of an A-minor is attributed to the stem or loop, and each pair of nucleotides is attributed to their relative position within the secondary structure. By analyzing classes of A-minors in known RNA structures, we found that the largest classes are mostly homogeneous and preferably localize with known A-minor co-motifs, e.g. tetraloop-tetraloop receptor and coaxial stacking. Detailed analysis of local A-minors within internal loops revealed a novel recurrent RNA tertiary motif, the across-bulged motif. Interestingly, the motif resembles the previously known GAAA/11nt motif but with the local adenines performing the role of the GAAA-tetraloop. By using machine learning, we show that particular classes of local A-minors can be predicted from sequence and secondary structure. The proposed classification is the first step toward automatic annotation of not only A-minors and their co-motifs but various types of RNA tertiary motifs as well.

Author Correction: Plants with genetically encoded autoluminescence.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Plants with genetically encoded autoluminescence.

Autoluminescent plants engineered to express a bacterial bioluminescence gene cluster in plastids have not been widely adopted because of low light output. We engineered tobacco plants with a fungal bioluminescence system that converts caffeic acid (present in all plants) into luciferin and report self-sustained luminescence that is visible to the naked eye. Our findings could underpin development of a suite of imaging tools for plants.

The presence of cross-ß-structure as a key determinant of carbonic anhydrase amyloid fibrils cytotoxicity.

In most cases high cytotoxicity is characteristic of aggregates formed during lag phase of amyloid formation, whereas mature fibrils represent the depot of protein molecules incapable of damaging cell membranes. However, new experimental data show that in cases of some proteins the fibrils are the most toxic type of aggregates. Meanwhile, structural characteristics of cytotoxic fibrils and mechanisms of their cell damaging action are insufficiently explored. This work is dedicated to studying amyloid aggregation of bovine carbonic anhydrase (BCA) and effect of aggregates formed at different stages of amyloid formation on viability of the cells. Here we demonstrate that oligomers formed during lag phase do not decrease cell viability, whereas protofibrils and amyloids of BCA are cytotoxic. Obtained results allow concluding that toxicity of BCA aggregates is associated with the presence of amyloid cross-ß-structure, which signature is absorbance peak at low wavenumbers at FTIR spectra (1615-1630 cm-1). Our data suppose that cross-ß-core of ВСА amyloid fibrils is responsible for their cytotoxicity.

Genetically encodable bioluminescent system from fungi.

Bioluminescence is found across the entire tree of life, conferring a spectacular set of visually oriented functions from attracting mates to scaring off predators. Half a dozen different luciferins, molecules that emit light when enzymatically oxidized, are known. However, just one biochemical pathway for luciferin biosynthesis has been described in full, which is found only in bacteria. Here, we report identification of the fungal luciferase and three other key enzymes that together form the biosynthetic cycle of the fungal luciferin from caffeic acid, a simple and widespread metabolite. Introduction of the identified genes into the genome of the yeast Pichia pastoris along with caffeic acid biosynthesis genes resulted in a strain that is autoluminescent in standard media. We analyzed evolution of the enzymes of the luciferin biosynthesis cycle and found that fungal bioluminescence emerged through a series of events that included two independent gene duplications. The retention of the duplicated enzymes of the luciferin pathway in nonluminescent fungi shows that the gene duplication was followed by functional sequence divergence of enzymes of at least one gene in the biosynthetic pathway and suggests that the evolution of fungal bioluminescence proceeded through several closely related stepping stone nonluminescent biochemical reactions with adaptive roles. The availability of a complete eukaryotic luciferin biosynthesis pathway provides several applications in biomedicine and bioengineering.