The Electrostatic Basis of Diacylglycerol Pyrophosphate—Protein Interaction

Diacylglycerol pyrophosphate (DGPP) is an anionic phospholipid formed in plants, yeast, and parasites under multiple stress stimuli. It is synthesized by the phosphorylation action of phosphatidic acid (PA) kinase on phosphatidic acid, a signaling lipid with multifunctional properties. PA functions in the membrane through the interaction of its negatively charged phosphomonoester headgroup with positively charged proteins and ions. DGPP, like PA, can interact electrostatically via the electrostatic-hydrogen bond switch mechanism but differs from PA in its overall charge and shape. The formation of DGPP from PA alters the physicochemical properties as well as the structural dynamics of the membrane. This potentially impacts the molecular and ionic binding of cationic proteins and ions with the DGPP enriched membrane. However, the results of these important interactions in the stress response and in DGPP’s overall intracellular function is unknown. Here, using 31P MAS NMR, we analyze the effect of the interaction of low DGPP concentrations in model membranes with the peptides KALP23 and WALP23, which are flanked by positively charged Lysine and neutral Tryptophan residues, respectively. Our results show a significant effect of KALP23 on the charge of DGPP as compared to WALP23. There was, however, no significant effect on the charge of the phosphomonoester of DGPP due to the interaction with positively charged lipids, dioleoyl trimethylammonium propane (DOTAP) and dioleoyl ethyl-phosphatidylcholine (EtPC). Divalent calcium and magnesium cations induce deprotonation of the DGPP headgroup but showed no noticeable differences on DGPP’s charge. Our results lead to a novel model for DGPP—protein interaction.

The Cryo-EM Structures of two Amphibian Antimicrobial Cross-β Amyloid Fibrils

The amyloid-antimicrobial link hypothesis is based on antimicrobial properties found in human amyloids involved in neurodegenerative and systemic diseases, along with amyloidal structural properties found in antimicrobial peptides (AMPs) across kingdoms of life. Supporting this hypothesis, we here determined the fibril structure of two AMPs from amphibians, uperin 3.5 and aurein 3.3, by cryogenic electron microscopy (cryo-EM), revealing amyloid cross-β fibrils of mated β-sheets at atomic resolution. Uperin 3.5 displayed substantial polymorphism with a protofilament of two mated β-sheets. The determined structure was a polymorph showing a 3-blade symmetrical propeller of nine peptides per fibril layer including tight β-sheet interfaces. This cross-β cryo-EM structure complements the cross-α fibril conformation previously determined by a crystal structure, substantiating a secondary structure switch mechanism of uperin 3.5. The aurein 3.3 arrangement consisted of six peptides per fibril layer, all showing kinked β-sheets allowing a rounded compactness of the fibril. The kinked β-sheets are similar to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked segments) found in human functional amyloids. The amyloidal properties of antimicrobial peptides shed light on a mechanism of regulation of animicrobial activity involving self-assembly and fibril morphological variations. Moreover, the known endurance of amyloid structures can provide a template for the design of sturdy antimicrobials.

FOXO1 Is a Critical Switch Molecule for Autophagy and Apoptosis of Sow Endometrial Epithelial Cells Caused by Oxidative Stress

Oxidative stress (OS) is involved in various reproductive diseases and can induce autophagy and apoptosis, which determine the different fates of cells. However, the sequence and the switch mechanism between autophagy and apoptosis are unclear. Here, we reported that chronic restraint stress (CRS) induced OS (decreased T-AOC, T-SOD, CAT and GSH-Px and increased MDA) and then disturbed the endocrine environment of sows during early pregnancy, including the hypothalamic-pituitary-ovarian (HPO) and the hypothalamic-pituitary-adrenal (HPA) axes. Meanwhile, after CRS, the KEAP1/NRF2 pathway was inhibited and attenuated the antioxidative ability to cause OS of the endometrium. The norepinephrine (NE) triggered β2-AR to activate the FOXO1/NF-κB pathway, which induced endometrial inflammation. CRS induced the caspase-dependent apoptosis pathway and caused MAP1LC3-II accumulation, SQSTM1/p62 degradation, and autophagosome formation to initiate autophagy. Furthermore, in vitro, a cellular OS model was established by adding hydrogen peroxide into cells. Low OS maintained the viability of endometrial epithelial cells by triggering autophagy, while high OS induced cell death by initiating caspase-dependent apoptosis. Autophagy preceded the occurrence of apoptosis, which depended on the subcellular localization of FOXO1. In the low OS group, FOXO1 was exported from the nucleus to be modified into Ac-FOXO1 and bound to ATG7 in the cytoplasm, which promoted autophagy to protect cells. In the high OS group, FOXO1 located in the nucleus to promote transcription of proapoptotic proteins and then induce apoptosis. Here, FOXO1, as a redox sensor switch, regulated the transformation of cell autophagy and apoptosis. In summary, the posttranslational modification of FOXO1 may become the target of OS treatment.

Competitive dynamics underlie cognitive improvements during sleep

We provide evidence that human sleep is a competitive arena in which cognitive domains vie for limited resources. Using pharmacology and effective connectivity analysis, we demonstrate that long-term memory and working memory are served by distinct offline neural mechanisms that are mutually antagonistic. Specifically, we administered zolpidem to increase central sigma activity and demonstrated targeted suppression of autonomic vagal activity. With effective connectivity, we determined the central activity has greater causal influence over autonomic activity, and the magnitude of this influence during sleep produced a behavioral trade-off between offline long-term and working memory processing. These findings suggest a sleep switch mechanism that toggles between central sigma-dependent long-term memory and autonomic vagal-dependent working memory processing.

Multi-Hop Question Generation Using Hierarchical Encoding-Decoding and Context Switch Mechanism

Neural auto-regressive sequence-to-sequence models have been dominant in text generation tasks, especially the question generation task. However, neural generation models suffer from the global and local semantic semantic drift problems. Hence, we propose the hierarchical encoding–decoding mechanism that aims at encoding rich structure information of the input passages and reducing the variance in the decoding phase. In the encoder, we hierarchically encode the input passages according to its structure at four granularity-levels: [word, chunk, sentence, document]-level. Second, we progressively select the context vector from the document-level representations to the word-level representations at each decoding time step. At each time-step in the decoding phase, we progressively select the context vector from the document-level representations to word-level. We also propose the context switch mechanism that enables the decoder to use the context vector from the last step when generating the current word at each time-step.It provides a means of improving the stability of the text generation process during the decoding phase when generating a set of consecutive words. Additionally, we inject syntactic parsing knowledge to enrich the word representations. Experimental results show that our proposed model substantially improves the performance and outperforms previous baselines according to both automatic and human evaluation. Besides, we implement a deep and comprehensive analysis of generated questions based on their types.

The spatial organization of replication is determined by cell size independently of chromosome copy number

We have studied how Escherichia coli coordinates replication and division cycles by tracking replisomes in individual cells through thousands of division cycles. By analysing the replication and division of the cells at various growth rates and with different genetic perturbations, we have reached four major conclusions: (1) Initiation is not based on titration of DnaA to the chromosomal binding sites because initiation is still coordinated with the division cycle several generations after DnaA synthesis is turned off. (2) We can rule out that initiation is triggered by a simple DnaA-ADP/ATP switch mechanism because initiation is still triggered at a well-defined cell size in a DARS1/DARS2/datA triple knockout if DnaA is overexpressed. (3) From the spatial organization of replisomes at different growth rates, it appears as if initiation is triggered by termination, but this hypothesis is ruled out by an NGS-based marker frequency analysis. (4) Overlapping replication cycles do not affect the spatial organization of the replication machinery and it appears as if each replication center contains a fixed number of DnaN molecules independently of how many replication forks are being processed.

The crystal structure of bromide-bound GtACR1 reveals a pre-activated state in the transmembrane anion tunnel

The crystal structure of the light-gated anion channel GtACR1 reported in our previous Research Article (Li et al., 2019) revealed a continuous tunnel traversing the protein from extracellular to intracellular pores. We proposed the tunnel as the conductance channel closed by three constrictions: C1 in the extracellular half, mid-membrane C2 containing the photoactive site, and C3 on the cytoplasmic side. Reported here, the crystal structure of bromide-bound GtACR1 reveals structural changes that relax the C1 and C3 constrictions, including a novel salt-bridge switch mechanism involving C1 and the photoactive site. These findings indicate that substrate binding induces a transition from an inactivated state to a pre-activated state in the dark that facilitates channel opening by reducing free energy in the tunnel constrictions. The results provide direct evidence that the tunnel is the closed form of the channel of GtACR1 and shed light on the light-gated channel activation mechanism.

The Essential and Enigmatic Role of ABC Transporters in Bt Resistance of Noctuids and Other Insect Pests of Agriculture

In the last ten years, ABC transporters have emerged as unexpected yet significant contributors to pest resistance to insecticidal pore-forming proteins from Bacillus thuringiensis (Bt). Evidence includes the presence of mutations in resistant insects, heterologous expression to probe interactions with the three-domain Cry toxins, and CRISPR/Cas9 knockouts. Yet the mechanisms by which ABC transporters facilitate pore formation remain obscure. The three major classes of Cry toxins used in agriculture have been found to target the three major classes of ABC transporters, which requires a mechanistic explanation. Many other families of bacterial pore-forming toxins exhibit conformational changes in their mode of action, which are not yet described for the Cry toxins. Three-dimensional structures of the relevant ABC transporters, the multimeric pore in the membrane, and other proteins that assist in the process are required to test the hypothesis that the ATP-switch mechanism provides a motive force that drives Cry toxins into the membrane. Knowledge of the mechanism of pore insertion will be required to combat the resistance that is now evolving in field populations of insects, including noctuids.

Activation mechanism of a small prototypic Rec-GGDEF diguanylate cyclase

Diguanylate cyclases synthesising the bacterial second messenger c-di-GMP are found to be regulated by a variety of sensory input domains that control the activity of their catalytical GGDEF domain, but how activation proceeds mechanistically is, apart from a few examples, still largely unknown. As part of two-component systems, they are activated by cognate histidine kinases that phosphorylate their Rec input domains. DgcR from Leptospira biflexa is a constitutively dimeric prototype of this class of diguanylate cyclases. Full-length crystal structures reveal that BeF3- pseudo-phosphorylation induces a relative rotation of two rigid halves in the Rec domain. This is coupled to a reorganisation of the dimeric structure with concomitant switching of the coiled-coil linker to an alternative heptad register. Finally, the activated register allows the two substrate-loaded GGDEF domains, which are linked to the end of the coiled-coil via a localised hinge, to move into a catalytically competent dimeric arrangement. Bioinformatic analyses suggest that the binary register switch mechanism is utilised by many diguanylate cyclases with N-terminal coiled-coil linkers.

The Assembly Switch Mechanism of FtsZ Filament Revealed by All-Atom Molecular Dynamics Simulations and Coarse-Grained Models

Bacterial cytoskeletal protein FtsZ binds and hydrolyzes GTP, and assembles into dynamic filaments that are essential for cell division. Here, we used a multi-scale computational strategy that combined all-atom molecular dynamics (MD) simulations and coarse-grained models to reveal the conformational dynamics of assembled FtsZ. We found that the top end of a filament is highly dynamic and can undergo T-to-R transitions in both GTP- and GDP-bound states. We observed several subcategories of nucleation related dimer species, which leading to a feasible nucleation pathway. In addition, we observed that FtsZ filament exhibits noticeable amounts of twisting, indicating a substantial helicity of the FtsZ filament. These results agree with the previously models and experimental data. Anisotropy network model (ANM) analysis revealed a polymerization enhanced assembly cooperativity, and indicated that the cooperative motions in FtsZ are encoded in the structure. Taken together, our study provides a molecular-level understanding of the diversity of the structural states of FtsZ and the relationships among polymerization, hydrolysis, and cooperative assembly, which should shed new light on the molecular basis of FtsZ’s cooperativity.