GSK1702934A and M085 directly activate TRPC6 via a mechanism of stimulating the extracellular cavity formed by the pore helix and transmembrane helix S6.

Transient receptor potential canonical (TRPC) channels, as important membrane proteins regulating intracellular calcium (Ca2+i) signaling, are involved in a variety of physiological and pathological processes. Activation and regulation of TRPC are more dependent on membrane or intracellular signals. However, how extracellular signals regulate TRPC6 function remains to be further investigated. Here, we suggest that two distinct small molecules, M085 and GSK1702934A, directly activate TRPC6, both through a mechanism of stimulation of extracellular sites formed by the pore helix (PH) and transmembrane (TM) helix S6. In silico docking scanning of TRPC6 identified three extracellular sites that can bind small molecules, of which only mutations on residues of PH and S6 helix significantly reduced the apparent affinity of M085 and GSK1702934A and attenuated the maximal response of TRPC6 to these two chemicals by altering channel gating of TRPC6. Combing metadynamics, molecular dynamics simulations, and mutagenesis, we revealed that W679, E671, E672, and K675 in the PH and N701 and Y704 in the S6 helix constitute an orthosteric site for the recognition of these two agonists. The importance of this site was further confirmed by covalent modification of amino acid residing at the interface of the PH and S6 helix. Given that three structurally distinct agonists M085, GSK1702934A, and AM-0883, act at this site, as well as the occupancy of lipid molecules at this position found in other TRP subfamilies, it is suggested that the cavity formed by the PH and S6 has an important role in the regulation of TRP channel function by extracellular signals.

Metabolic engineering of Yarrowia lipolytica for improving squalene production.

The aim of this present research is to enhance the squalene production in Yarrowia lipolytica using pathway engineering and bioprocess engineering. Firstly, to improve the production of squalene, the endogenous HMG-CoA reductase (HMG1) was overexpressed in Y. lipolytica to yield 208.88 mg/L squalene. Secondly, the HMG1 and diacylglycerol acyltranferase (DGA1) were co-overexpressed, the derived recombinant Y. lipolytica SQ-1 strain produced 439.14 mg/L of squalene. Thirdly, by optimizing the fermentation medium, the improved titer of squalene with 514.34 mg/L was obtained by the engineered strain SQ-1 grown on YPD-80 medium. Finally, by optimizing the addition concentrations of acetate, citrate and terbinafine, the 731.18 mg/L squalene was produced in the engineered strain SQ-1 with the addition of 0.5 mg/L terbinafine. This work describes the highest reported squalene titer in Y. lipolytica to date. This study will provide the foundation for further engineering Y. lipolytica capable of cost-efficiently producing squalene.

[Advances in the study of plant microRNAs].

Plant microRNAs (miRNAs) are single-stranded RNA molecules of around 22 nucleotides (nt) in length that are associated with the RNA-induced silencing complex (RISC). They act as post-transcriptional negative regulators of gene expression mainly by guiding cleavage or attenuating the translation of target transcripts. The targets of plant miRNAs often belong to transcription factors families involved in the control of developmental processes and defense responses. In the present paper, we reviewed the recent advances in our understanding of the biogenesis and mechanism of action of plant miRNAs, as well as the regulatory roles in plants.