FLOWERING BHLH transcriptional activators control expression of the photoperiodic flowering regulator CONSTANS in Arabidopsis.

Many plants monitor day-length changes throughout the year and use the information to precisely regulate the timing of seasonal flowering for maximum reproductive success. In Arabidopsis thaliana, transcriptional regulation of the CONSTANS (CO) gene and posttranslational regulation of CO protein are crucial mechanisms for proper day-length measurement in photoperiodic flowering. Currently, the CYCLING DOF FACTOR proteins are the only transcription factors known to directly regulate CO gene expression, and the mechanisms that directly activate CO transcription have remained unknown. Here we report the identification of four CO transcriptional activators, named FLOWERING BHLH 1 (FBH1), FBH2, FBH3, and FBH4. All FBH proteins are related basic helix-loop-helix-type transcription factors that preferentially bind to the E-box cis-elements in the CO promoter. Overexpression of all FBH genes drastically elevated CO levels and caused early flowering regardless of photoperiod, whereas CO levels were reduced in the fbh quadruple mutants. In addition, FBH1 is expressed in the vascular tissue and bound near the transcription start site of the CO promoter in vivo. Furthermore, FBH homologs in poplar and rice induced CO expression in Arabidopsis. These results indicate that FBH proteins positively regulate CO transcription for photoperiodic flowering and that this mechanism may be conserved in diverse plant species. Our results suggest that the diurnal CO expression pattern is generated by a concert of redundant functions of positive and negative transcriptional regulators.

De novo design of ACE2 protein decoys to neutralize SARS-CoV-2.

There is an urgent need for the ability to rapidly develop effective countermeasures for emerging biological threats, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a generalized computational design strategy to rapidly engineer de novo proteins that precisely recapitulate the protein surface targeted by biological agents, like viruses, to gain entry into cells. The designed proteins act as decoys that block cellular entry and aim to be resilient to viral mutational escape. Using our novel platform, in less than ten weeks, we engineered, validated, and optimized de novo protein decoys of human angiotensin-converting enzyme 2 (hACE2), the membrane-associated protein that SARS-CoV-2 exploits to infect cells. Our optimized designs are hyperstable de novo proteins (∼18-37 kDa), have high affinity for the SARS-CoV-2 receptor binding domain (RBD) and can potently inhibit the virus infection and replication in vitro. Future refinements to our strategy can enable the rapid development of other therapeutic de novo protein decoys, not limited to neutralizing viruses, but to combat any agent that explicitly interacts with cell surface proteins to cause disease.

De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2.

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo human angiotensin-converting enzyme 2 (hACE2) decoys to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The best monovalent decoy, CTC-445.2, bound with low nanomolar affinity and high specificity to the receptor-binding domain (RBD) of the spike protein. Cryo-electron microscopy (cryo-EM) showed that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, showed ~10-fold improvement in binding. CTC-445.2d potently neutralized SARS-CoV-2 infection of cells in vitro, and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge.