Li M et al. (2007) Biochemistry "Phosphorylation and functions of inhibitor-2 family of proteins."

Li M, Brautigan DL, Satinover DL

[Biochemistry, 2007]

Protein phosphatase-1 (PP1) is an essential protein Ser/Thr phosphatase that is extraordinarily conserved from yeast to human, and Inhibitor-2 (I-2) is the most ancient of the heat-stable proteins specific for PP1. We identified novel I-2 homologues in Caenorhabditis elegans (Ce) and Xenopus laevis (Xe) and compared them to the I-2 proteins from Homo sapiens (Hs), Saccharomyces cerevisiae (GLC8), and Drosophila melanogaster (Dm). The Ce I-2 and Dm I-2 showed the highest potency inhibition of rabbit PP1 with IC50 near 5 nM compared to Hs I-2 and Xe I-2 with IC50 between 10 and 50 nM and GLC8 with >100-fold lower activity. Inhibition of PP1 bound to Nek2 kinase activated the kinase to phosphorylate a C-Nap1 domain substrate. All the species of I-2 except GLC8 activated the Nek2::PP1 to the same extent as microcystin-LR. Only Hs I-2 and Xe I-2, not the I-2 proteins more divergent in sequence, directly activated human Aurora-A kinase. Various species of I-2 have a common PxTP phosphorylation site that showed a wide range of reactivity with GSK3, ERK, or CDC2/cyclinB1 kinases. The Suc1 subunit of CDC2/cyclinB1 enhanced reactivity with I-2, consistent with this being a site of mitotic phosphorylation. The results show species specificity among the I-2 family within the context of conserved PP1 inhibitory activity and variable phosphorylation by Pro-directed kinases.

Li M et al. (2017) Nature "Structure of a eukaryotic cyclic-nucleotide-gated channel."

Zhou X, Yang J, Gong Y, Li M, Michailidis I, Su D, Li H, Wang S, Li X

[Nature, 2017]

Cyclic-nucleotide-gated channels are essential for vision and olfaction. They belong to the voltage-gated ion channel superfamily but their activities are controlled by intracellular cyclic nucleotides instead of transmembrane voltage. Here we report a 3.5-A-resolution single-particle electron cryo-microscopy structure of a cyclic-nucleotide-gated channel from Caenorhabditis elegans in the cyclic guanosine monophosphate (cGMP)-bound open state. The channel has an unusual voltage-sensor-like domain, accounting for its deficient voltage dependence. A carboxy-terminal linker connecting S6 and the cyclic-nucleotide-binding domain interacts directly with both the voltage-sensor-like domain and the pore domain, forming a gating ring that couples conformational changes triggered by cyclic nucleotide binding to the gate. The selectivity filter is lined by the carboxylate side chains of a functionally important glutamate and three rings of backbone carbonyls. This structure provides a new framework for understanding mechanisms of ion permeation, gating and channelopathy of cyclic-nucleotide-gated channels and cyclic nucleotide modulation of related channels.

Li M et al. (2016) Bioengineered "Modeling the thermotaxis behavior of C.elegans based on the artificial neural network."

Deng X, Tang Y, Wang J, Chen Q, Li M

[Bioengineered, 2016]

This research aims at modeling the thermotaxis behavior of C.elegans which is a kind of nematode with full clarified neuronal connections. Firstly, this work establishes the motion model which can perform the undulatory locomotion with turning behavior. Secondly, the thermotaxis behavior is modelled by nonlinear functions and the nonlinear functions are learned by artificial neural network. Once the artificial neural networks have been well trained, they can perform the desired thermotaxis behavior. Last, several testing simulations are carried out to verify the effectiveness of the model for thermotaxis behavior. This work also analyzes the different performances of the model under different environments. The testing results reveal the essence of the thermotaxis of C.elegans to some extent, and theoretically support the research on the navigation of the crawling robots.

Li M et al. (2024) Talanta "An "off-on" fluorescent probe for imaging pyruvic acid in living systems."

Liu C, Chen L, Li X, Li M

[Talanta, 2024]

Pyruvic acid (PA) is an α-keto acid which exert important biological and pathological functions. The current PA profiling assays are mainly based on the ultraviolet spectroscopy and electrochemical biosensor, requiring killing cells and destroying tissues which limit their application in living cells. Optical imaging provides nondestructive powerful and detective tools to better understand the physiological and pathological role of PA in living systems. However, as far as we know, none of"off - on" PA fluorescent sensor has been developed. Herein, we reported a PA recognition reaction that arylhydroxylamine group could be selectively reduced to acetylamide group by PA. With this recognition reaction, a fluorescence probe (FPA) based on the photoinduced electron transfer (PET) pathway was designed, synthesized and could release strong fluorescence at 447 nm. We proved that FPA could detect PA in aqueous solution, living cells, Caenorhabditis elegans and the roots of Arabidopsis thaliana with good selectivity and sensitivity as low as 0.42 μM. In addition, we successfully using probe FPA to study the intracellular PA production pathway in cells and evaluated its physiological level in Arabidopsis roots at different growth stages. The results show that the physiological level of PA in Arabidopsis thaliana roots is closely associated with their growth stages, which indicated that PA might act as a carbon source and related growth signaling molecule to promote plant growth and root elongation. Therefore, we expect probe FPA to be a powerful tool to better understand the physiological and pathological role of PA.

Li M et al. (2017) BMC Microbiol "Lactobacillus-derived extracellular vesicles enhance host immune responses against vancomycin-resistant enterococci."

Lee K, Li M, Hsu M, Nau G, Mylonakis E, Ramratnam B

[BMC Microbiol, 2017]

BACKGROUND: Probiotic bacteria are known to modulate host immune responses against various pathogens. Recently, extracellular vesicles (EVs) have emerged as potentially important mediators of host-pathogen interactions. In this study, we explored the role of L. plantarum derived EVs in modulating host responses to vancomycin-resistant Enterococcus faecium (VRE) using both Caenorhabditis elegans and human cells. RESULTS: Our previous work has shown that probiotic conditioning C. elegans with L. acidophilus NCFM prolongs the survival of nematodes exposed to VRE. Similarly, L. plantarum WCFS1 derived extracellular vesicles (LDEVs) also significantly protected the worms against VRE infection. To dissect the molecular mechanisms of this EV-induced protection, we found that treatment of C. elegans with LDEVs significantly increased the transcription of host defense genes, cpr-1 and clec-60. Both cpr-1 and clec-60 have been previously reported to have protective roles against bacterial infections. Incubating human colon-derived Caco-2 cells with fluorescent dye-labeled LDEVs confirmed that LDEVs could be transported into the mammalian cells. Furthermore, LDEV uptake was associated with significant upregulation of CTSB, a human homologous gene of cpr-1, and REG3G, a human gene that has similar functions to clec-60. CONCLUSIONS: We have found that EVs produced from L. plantarum WCFS1 up-regulate the expression of host defense genes and provide protective effects on hosts. Using probiotic-derived EVs instead of probiotic bacteria themselves, this study provides a new direction to treat antimicrobial resistant pathogens, such as VRE.

Li M et al. (2013) Anal Chem "Stable isotope probing and Raman spectroscopy for monitoring carbon flow in a food chain and revealing metabolic pathway."

Jousset A, Gibson CM, Li M, Huang WE, Fowler PW

[Anal Chem, 2013]

Accurately measuring carbon flows is a challenge for understanding processes such as diverse intracellular metabolic pathways and predator-prey interactions. Combined with stable isotope probing (SIP), single-cell Raman spectroscopy was demonstrated for the first time to link the food chain from carbon substrate to bacterial prey up to predators at the single-cell level in a quantitative and nondestructive manner. Escherichia coli OP50 with different (13)C content, which were grown in a mixture of (12)C- and fully carbon-labeled (13)C-glucose (99%) as a sole carbon source, were fed to the nematode. The (13)C signal in Caenorhabditis elegans was proportional to the (13)C content in E. coli. Two Raman spectral biomarkers (Raman bands for phenylalanine at 1001 cm(-1) and thymine at 747 cm(-1) Raman bands), were used to quantify the (13)C content in E. coli and C. elegans over a range of 1.1-99%. The phenylalanine Raman band was a suitable biomarker for prokaryotic cells and thymine Raman band for eukaryotic cells. A biochemical mechanism accounting for the Raman red shifts of phenylalanine and thymine in response to (13)C-labeling is proposed in this study and is supported by quantum chemical calculation. This study offers new insights of carbon flow via the food chain and provides a research tool for microbial ecology and investigation of biochemical pathways.

Li M et al. (2019) Cell Metab "Transient Receptor Potential V Channels Are Essential for Glucose Sensing by Aldolase and AMPK."

Zong Y, Xie C, Ma T, Lin SY, Hardie DG, Liu QF, Xie XS, Liu Y, Ye Z, Jiang D, Wu YQ, Zhang C, Zhang CS, Piao HL, Chen J, Yang Y, Li Y, Cui J, Yu L, Tian X, Feng JW, Wang W, Lin SC, Li M, Lin Z, Li X, Zhou Z, Wu J, Chen X, Wu Y, Hu M

[Cell Metab, 2019]

Fructose-1,6-bisphosphate (FBP) aldolase links sensing of declining glucose availability to AMPK activation via the lysosomal pathway. However, how aldolase transmits lack of occupancy by FBP to AMPK activation remains unclear. Here, we show that FBP-unoccupied aldolase interacts with and inhibits endoplasmic reticulum (ER)-localized transient receptor potential channel subfamily V, inhibiting calcium release in low glucose. The decrease of calcium at contact sites between ER and lysosome renders the inhibited TRPV accessible to bind the lysosomal v-ATPase that then recruits AXIN:LKB1 to activate AMPK independently of AMP. Genetic depletion of TRPVs blocks glucose starvation-induced AMPK activation in cells and liver of mice, and in nematodes, indicative of physical requirement of TRPVs. Pharmacological inhibition of TRPVs activates AMPK and elevates NAD⁺ levels in aged muscles, rejuvenating the animals' running capacity. Our study elucidates that TRPVs relay the FBP-free status of aldolase to the reconfiguration of v-ATPase, leading to AMPK activation in low glucose.

Li M et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Metabolic Arrest Confers Resistance Against the Fumigant Phosphine"

Tuck A, Li M, Ebert P R, Schirra H J, Cheung J

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

The fumigant, phosphine, is a metabolic toxin that is used to protect stored grain from insect pests. The phosphine resistant mutant, pre-33, has a basal metabolic rate only 20% that of the wild type strain, N2. Upon exposure to phosphine, TCA cycle genes are suppressed from normal expression levels in pre-33, indicating that not only does the mutant exhibit a basal suppression of metabolism, but that the metabolic capacity can be depressed further, and by a different mechanism, upon exposure to phosphine at 500 ppm. Further evidence of metabolic disruption in the mutant is that it is completely resistant to synergistic enhancement of phosphine toxicity under hyperoxic conditions. Metabolite profiling by NMR identifies metabolic disruption, specifically accumulation of metabolites that feed into the TCA cycle, as a signature of phosphine toxicity, but not resistance. In addition to basal suppression of metabolism and further suppression of TCA cycle genes by pre-33 upon sublethal exposure to phosphine at 500 ppm, the unfolded protein response is also strongly induced. None of these phenotypes occur in N2 nematodes treated at 500 ppm, nor do they occur in pre-33 treated at 4000 ppm, which results in a similar level of mortality. Thus, arrested metabolism and induction of a protein maintenance response is characteristic of pre-33 nematodes treated with a dose of phosphine that, while sublethal for pre-33, would kill wild type, N2, nematodes. The metabolic response to phosphine exposure was also tested in yeast grown in either glucose medium that allowed fermentation, or in glycerol medium that forced the yeast to respire aerobically. Fermentation resulted in yeast that were completely resistant to phosphine toxicity. Interestingly, exposure to phosphine during aerobic respiration resulted in the complete growth arrest of the yeast, but did not result in any mortality. This result suggests that metabolic arrest is a general mechanism of phosphine resistance in aerobically respiring organisms.

Li M et al. (2005) Dev Cell "Regulatory mutations of mir-48, a C. elegans let-7 family MicroRNA, cause developmental timing defects."

Lau NC, Rougvie AE, Jones-Rhoades MW, Li M, Bartel DP

[Dev Cell, 2005]

The C. elegans heterochronic genes program stage-specific temporal identities in multiple tissues during larval development. These genes include the first two miRNA-encoding genes discovered, lin-4 and let-7. We show that lin-58 alleles, identified as lin-4 suppressors, define another miRNA that controls developmental time. These alleles are unique in that they contain point mutations in a gene regulatory element of mir-48, a let-7 family member. mir-48 is expressed prematurely in lin-58 mutants, whereas expression of mir-241, another let-7 family member residing immediately upstream of mir-48, appears to be unaffected. A mir-48 transgene bearing a lin-58 point mutation causes strong precocious phenotypes in the hypodermis and vulva when expressed from multicopy arrays. mir-48::gfp fusions reveal expression in these tissues, and inclusion of a lin-58 mutation causes precocious and enhanced gfp expression. These results suggest that lin-58 alleles disrupt a repressor binding site that restricts the time of miR-48 action in wild-type animals.

Li M et al. (2024) Nat Commun "Structural basis for C. elegans pairing center DNA binding specificity by the ZIM/HIM-8 family proteins."

Shi Y, Zhu C, Zhang Y, Han X, Wang S, Guang S, Hou X, Li F, Lv M, Xu M, Kuang Y, Liu Y, Xu Z, Huang X, Li M

[Nat Commun, 2024]

Pairing center (PC) on each chromosome of Caenorhabditis elegans is crucial for homolog pairing and initiating synapsis. Within each PC, clusters of 11/12 bp DNA motif recruit one of four paralogous meiosis-specific proteins: ZIM-1, ZIM-2, ZIM-3, or HIM-8. However, the mechanistic basis underlying the specificity of ZIM/HIM-8-PC DNA interaction remains elusive. Here, we describe crystal structures of HIM-8, ZIM-1 and ZIM-2 DNA binding domains (ZF1, ZF2 and CTD) in complex with their cognate PC DNA motifs, respectively. These structures demonstrated the ZF1-2-CTD folds as an integrated structural unit crucial for its DNA binding specificity. Base-specific DNA-contacting residues are exclusively distributed on ZF1-2 and highly conserved. Furthermore, the CTD potentially contributes to the conformational diversity of ZF1-2, imparting binding specificity to distinct PC DNA motifs. These findings shed light on the mechanism governing PC DNA motif recognition by ZIM/HIM-8 proteins, suggesting a co-evolution relationship between PC DNA motifs and ZF1-2-CTD in shaping the specific recognition.