bioRxiv Science⌕ Search

Biology subjects

Ouyang, Q.

Publications and source records attributed to Ouyang, Q..

29 records · Page 2Linked to original sources

Plant-on-Chip: core morphogenesis processes in the tiny plant Wolffia australiana

A plant can be thought of as a colony comprising numerous growth buds, each developing to its own rhythm. Such lack of synchrony impedes efforts to describe core principles of plant morphogenesis, dissect the underlying mechanisms, and identify regulators. Here, we use the tiniest known angiosperm to overcome this challenge and provide an ideal model system for plant morphogenesis. We present a detailed morphological description of the monocot Wolffia australiana, as well as high-quality genome information. Further, we developed the Plant-on-Chip culture system and demonstrate the application of advanced technologies such as snRNA-seq, protein structure prediction, and gene editing. We provide proof-of-concept examples that illustrate how W. australiana can open a new horizon for deciphering the core regulatory mechanisms of plant morphogenesis. SignificanceWhat is the core morphogenetic process in angiosperms, a plant like a tree indeterminately growing, or a bud sequentially generating limited types of organs? Wolffia australiana, one of the smallest angiosperms in the world may help to make a distinction. Wolffia plantlet constitutes of only three organs that are indispensable to complete life cycle: one leaf, one stamen and one gynoecium. Before the growth tip is induced to flower, it keeps branching from the leaf axil and the branches separate from the main plantlet. Here we present a high-quality genome of W. australiana, detailed morphological description, a Plant-on-Chip cultural system, and some principle-proof experiments, demonstrating that W. australiana is a promising model system for deciphering core developmental program in angiosperms.

plant biology↗

A cooperative switch within the KaiC hexamer revealed by cryo-EM

The circadian clock of cyanobacteria is based on an approximately 24h rhythm in the phosphorylation level of KaiC, a hexameric ATPase. This oscillation can be reconstituted in vitro by incubating three proteins together with ATP. Like all chemical oscillators, this system must include a nonlinear, or switch-like, feedback loop, whose nature has been unclear. Here, by using single particle cryo-EM at near-atomic resolution we identified two major conformational states of KaiC subunits, denoted as the exposed state and the buried state, which may provide a structural basis of how the KaiC hexamer changes its conformation during the (day-night) phosphorylation-dephosphorylation cycle. We classify the abundance and pattern of exposed and buried states within hexamers for more than 160,000 KaiC particles. The statistics of the spatial arrangement of the two states in hexamers can be quantitatively fit by a simple statistical physics model with an interaction energy between neighboring subunits and a local field that depends on phosphorylation state. Our study shows that phosphorylation shifts the probability of each conformation and reveals that there is substantial cooperativity between neighboring subunits, which can allow a KaiC hexamer to respond in an ultrasensitive, switch-like manner to changes in the phosphorylation level.

biophysics↗

Scaling between DNA and cell size governs bacterial growth homeostasis and resource allocation

Bacteria maintain a stable cell size and a certain DNA content through proliferation as described by classic growth laws. How cells behave when this inherent scaling is broken, however, has rarely been interrogated. Here we engineered Escherichia coli cells with extremely low DNA contents using a tunable synthetic tool CRISPRori that temporarily inhibited chromosome replication initiation. A detailed mechanistic model coupling DNA replication, cell growth, and division revealed a fundamental DNA-centric growth law, which was validated by two observations. First, lineage dynamics were robust to large CRISPRori perturbations with division cycles rapidly restoring through a timer mechanism rather than the adder rule. Second, cellular growth transitioned into a linear regime at low DNA-cytoplasm ratios. Experiments and theory showed that in this regime, cellular resource was redirected to plasmid-borne gene expression. Together with the ability of CRISPRori to bi-directionally modulate plasmid copy numbers, these findings suggest a novel strategy for bio-production enhancement.

systems biology↗

Yeast cells under glucose-limitation environment need increased response cost for osmostress defense

Cells always make responses to environmental changes, involving dynamic expression of tens to hundreds of proteins. This response system may demand substantial cost and thus affect cell growth. Here, we studied the cells responses to osmostress under glucose-limitation environments. Through analyzed thirteen osmotic-downstream proteins and two related transcription factors, we found that the cells required stronger responses under low glucose concentrations than normal glucose condition after being stimulated by osmostress, even the cell growth rate was unchanged in these two constant conditions. We proposed and verified that under a glucose-limitation environment, the glycolysis intermediates were limited (defense reserve saving), which caused that cells needed more glycerol production enzymes to adapt to the osmostress. Further experiments proved that this defense reserve-saving strategy required cells to spend more response cost when facing stress, which on the other hand, enhanced the fitness for the coming environment variations via protein accumulation reserve.

systems biology↗

Compound- and fiber type-selective requirement of AMPKγ3 for insulin-independent glucose uptake in skeletal muscle

Objective: The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPK{gamma}3 isoform is uniquely expressed in skeletal muscle and also potentially in BAT. Here, we investigated the role that AMPK{gamma}3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed if {gamma}3 plays a role in adipose thermogenesis and browning. Methods: Global AMPK{gamma}3 knockout (KO) mice were generated. A systematic whole-body, tissue and molecular phenotyping linked to glucose homeostasis was performed in {gamma}3 KO and wild type (WT) mice. Glucose uptake in glycolytic and oxidative skeletal muscle ex vivo, as well as blood glucose clearance in response to small molecule AMPK activators that target nucleotide-binding domain of {gamma} subunit (AICAR) and allosteric drug and metabolite (ADaM) site located at the interface of the and {beta} subunit (991, MK-8722) were assessed. Oxygen consumption, thermography, and molecular phenotyping with a {beta}3-adrenergic receptor agonist (CL-316,243) treatment were performed to assess BAT thermogenesis, characteristics and function. Results: Genetic ablation of {gamma}3 did not affect body weight, body composition, physical activity, and parameters associated with glucose homeostasis under chow or high fat diet. {gamma}3 deficiency had no effect on fiber-type composition, mitochondrial content and components, or insulin-stimulated glucose uptake in skeletal muscle. Glycolytic muscles in {gamma}3 KO mice showed a partial loss of AMPK2 activity, which was associated with reduced levels of AMPK2 and {beta}2 subunit isoforms. Notably, {gamma}3 deficiency resulted in a selective loss of AICAR-, but not MK-8722-induced blood glucose-lowering in vivo and glucose uptake specifically in glycolytic muscle ex vivo. We detected {gamma}3 in BAT and found that it preferentially interacts with 2 and {beta}2. We observed no differences in oxygen consumption, thermogenesis, morphology of BAT and inguinal white adipose tissue (iWAT), or markers of BAT activity between WT and {gamma}3 KO mice. Conclusions: These results demonstrate that {gamma}3 plays a key role in mediating AICAR- but not ADaM site binding drug-stimulated blood glucose clearance and glucose uptake specifically in glycolytic skeletal muscle. We also showed that {gamma}3 is dispensable for thermogenesis and browning of iWAT.

physiology↗

Unique intramolecular oxidative rearrangement N-nitrosation mechanism of non-heam iron-containing enzyme SznF

Non-heam iron-dependent enzyme SznF catalyzes a critical step of the L-arginine derived guanidine group rearrangement to produce the N-nitrosourea pharmacophore in the process of SZN biosynthesis. The intramolecular oxidative rearrangement process is accomplished in the Fe(II)-containing active site located at the cupin domain of SznF, with which the catalytic mechanism remains elusive. In this work, density functional theory methods have been employed to investigate possible catalytic mechanisms of SznF. The N-nitrosation reaction in SznF was found to follow an energetically favorable pathway which includes six consecutive steps: (1) formation of FeII-superoxo species with dioxgen binding on the iron center; (2) superoxo group attacking on the C{varepsilon} of substrate to form the peroxo-bridge complex; (3) C{varepsilon}-N{omega} bond homolysis to release N{omega}O; (4) peroxo bridge heterolytic cleavage; (5) deprotonation of [Formula] by Fe-O group; (6) the [Formula] couples with the N{omega}O group and generates the N-nitroso product. The reaction proceeds in an unexpected way during which the electrons shuttle among two NO groups of the substrate and the peroxo moiety to promote C{varepsilon}-N{omega} bond homolysis and O-O bond heterolysis sequentially without generating high-valent Fe-O species, which is distinct from any known reactions catalyzed by the iron-containing enzyme. The unusual mechanism of SznF shed light on the area of enzymatic N-nitrosation reactions.

biochemistry↗

Interactive regulation between aliphatic hydroxylation and aromatic hydroxylation of thaxtomin D in TxtC, a theoretical investigation

TxtC is an unusual bifunctional cytochrome P450 that is able to perform sequential aliphatic and aromatic hydroxylation of the diketopiperazine substrate thaxtomin D in two remote sites to produce thaxtomin A. Though the X-ray structure of TxtC complexed with thaxtomin D revealed a binding mode for its aromatic hydroxylation, the preferential hydroxylation site is aliphatic C14. It is thus intriguing to unravel how TxtC accomplishes such two-step catalytic hydroxylation on distinct aliphatic and aromatic carbons and why the aliphatic site is preferred in the hydroxylation step. In this work, by employing molecular docking and molecular dynamics (MD) simulation, we revealed that thaxtomin D could adopt two different conformations in the TxtC active site, which were equal in energy with either the aromatic C-H or aliphatic C14-H laying towards the active Cpd I oxyferryl moiety. Further ONIOM calculations indicated that the energy barrier for the rate-limiting hydroxylation step on the aliphatic C14 site was 8.9 kcal/mol more favorable than that on the aromatic C20 site. The hydroxyl group on the monohydroxylated intermediate thaxtomin B C14 site formed hydrogen bonds with Ser280 and Thr385, which induced the L-Phe moiety to rotate around the C{beta}-C{gamma} bond of the 4-nitrotryptophan moiety. Thus, it adopted an energy favorable conformation with aromatic C20 adjacent to the oxyferryl moiety. In addition, the hydroxyl group induced solvent water molecules to enter the active site, which propelled thaxtomin B towards the heme plane and resulted in heme distortion. Based on this geometrical layout, the rate-limiting aromatic hydroxylation energy barrier decreased to 15.4 kcal/mol, which was comparable to that of the thaxtomin D aliphatic hydroxylation process. Our calculations indicated that heme distortion lowered the energy level of the lowest Cpd I -vacant orbital, which promoted electron transfer in the rate-limiting thaxtomin B aromatic hydroxylation step in TxtC.

biochemistry↗

Yeast replicative aging leads to permanent cell cycle arrest in G1 effectuated by the start repressor Whi5

Yeast replicative aging has been a canonical model for aging research. Since replicative aging eventually leads to permanent cell cycle arrest, a fundamental question is how cells sense the signals from aging and communicate that to the cell cycle control machineries. Using microfluidic devices to track individual mother cells labeled by two different cell cycle markers Whi5-tdTomato and Myo1-EGFP, we measured the length of different cell cycle phases as a function of age and the distribution of cell death in different cell cycle phases. We found that the majority of the cells died in the G1 phase, and their G1 cell cycle length increased drastically in the last few cell divisions. This increase of G1 length correlates with the increase of the nuclear concentration of Whi5, which is a major transcriptional suppressor of the cell cycle start check point. Interestingly, this correlation is apparent only above a threshold concentration of Whi5. We show that in response to external stress, Whi5 concentration increases and cell growth slows down in a Whi5 dependent manner, and that Whi5 deletion significantly extends the lifespan. Together these data suggest the existence of a programmed control to arrest cell cycle in G1 in response to stress signals due to aging, and that Whi5 is a major mediator of this process. Our findings may have important implications in understanding senescence and cancer in mammalian cells, which have a parallel G1/S control system with Rb (a well known tumor suppressor) as the analog of Whi5.\n\nSignificance statementIn this work, we used microfluidic devices to track individual mother cells labeled by two cell cycle markers Whi5-tdTomato and Myo1-EGFP. We found that aging leads to significant lengthening of G1 phase in old cells and the eventual permanent cell cycle arrest in G1, and Whi5 plays an important role in implementing such a program. We show that oxidative stress can lead to the increase of Whi5 expression and the slow-down of cell division. Furthermore, Whi5 deletion significantly extends the lifespan. The result suggest the existence of a programmed control to arrest cell cycle in G1 in response to stress signals due to aging, and that Whi5 is a major mediator of this process.

cell biology↗

Nucleotide-Driven Triple-State Remodeling Of The AAA-ATPase Channel In The Activated Human 26S Proteasome

The proteasome is a sophisticated ATP-dependent molecular machine responsible for protein degradation in all eukaryotic cells. It remains elusive how conformational changes of the AAA-ATPase unfoldase in the regulatory particle (RP) control the gating of substrate-translocation channel to the proteolytic chamber of the core particle (CP). Here we report three alternative states of the ATP-{gamma}S-bound human proteasome, in which the CP gate is asymmetrically open, visualized by cryo-EM at near-atomic resolutions. Only four nucleotides are stably bound to the AAA-ATPase ring in the open-gate states. Concerted nucleotide exchange gives rise to a back-and-forth wobbling motion of the AAA-ATPase channel, coincident with remarkable transitions of their pore loops between the spiral staircase and saddle-shaped circle topologies. Gate opening in the CP is thus controlled with nucleotide-driven remodeling of the AAA-ATPase unfoldase. These findings demonstrate an elegant mechanism of allosteric coordination among sub-machines within the holoenzyme that is crucial for substrate translocation.

biochemistry↗

Conformational landscape of the p28-bound human proteasome regulatory particle

The proteasome holoenzyme is activated by its regulatory particle (RP) consisting of two subcomplexes, the lid and the base. A key event in base assembly is the formation of a heterohexameric ring of AAA-ATPases, which is guided by at least four RP assembly chaperones in mammals: PAAF1, p28/gankyrin, p27/PSMD9 and S5b. We determined a cryo-EM structure of the human RP in complex with its assembly chaperone p28 at 4.5-[A] resolution. The Rpn1-p28-AAA subcomplex in the p28-bound RP is highly dynamic and was resolved to subnanometer resolution in seven states, which recapitulate the conformational landscape of the complex. Surprisingly, the p28-bound AAA ring does not form a channel in the free RP. Instead, it spontaneously samples multiple open and closed topologies. Our analysis suggests that p28 guides the proteolytic core particle to select certain conformation of the ATPase ring for RP engagement in the last step of the chaperone-mediated proteasome assembly.

biochemistry↗

Phosphorylation energy and nonlinear kinetics as key determinants for G2/M transition in fission yeast cell cycle

The living cell is an open nonequilibrium biochemical system, where ATP hydrolysis serves as the energy source for a wide range of intracellular processes including the assurance for decision-making. In the fission yeast cell cycle, the transition from G2 phase to M phase is triggered by the activation of Cdc13/Cdc2 and Cdc25, and the deactivation of Wee1. Each of these three events involves a phosphorylation-dephosphorylation (PdP) cycle, and together they form a regulatory circuit with feedback loops. Almost all quantitative models for cellular networks in the past have invalid thermodynamics due to the assumption of irreversible enzyme kinetics. We constructed a thermodynamically realistic kinetic model of the G2/M circuit, and show that the phosphorylation energy ({Delta}G), which is determined by the cellular ATP/ADP ratio, critically controls the dynamics and the bistable nature of Cdc2 activation. Using fission yeast nucleoplasmic extract (YNPE), we are able to experimentally verify our model prediction that increased {Delta}G, being synergistic to the accumulation of Cdc13, drives the activation of Cdc2. Furthermore, Cdc2 activation exhibits bistability and hysteresis in response to changes in phosphorylation energy. These findings suggest that adequate maintenance of phosphorylation energy ensures the bistability and robustness of the activation of Cdc2 in the G2/M transition. Free energy might play a widespread role in biological decision-making processes, connecting thermodynamics with information processing in biology.

systems biology↗