Anti-inflammatory mechanisms of neutrophil membrane-coated nanoparticles without drug loading.

Neutrophil membrane-coated nanoparticles (NM-NPs) are nanomedicines with traits of mimicking the surface properties and functions of neutrophils, which are the most abundant type of white blood cells in the human body. NM-NPs have been widely used as targeted drug delivery systems for various inflammatory diseases, but their intrinsic effects on inflammation are not fully characterized yet. This study found that NM-NPs could modulate inflammation by multiple mechanisms without drug loading. NM-NPs could inhibit the recruitment of neutrophils and macrophages to the inflamed site by capturing chemokines and blocking their adhesion to inflamed endothelial cells. After internalized by macrophages and other phagocytic cells, NM-NPs could alter their phenotype by phosphatidylserine and simultaneously degrade the sequestered and neutralized cytokines and chemokines by lysosomal degradation. Under these effects, NM-NPs exhibited significant anti-inflammatory effects on LPS-induced inflammatory liver injury in vivo without drug loading. Our study unveiled the anti-inflammatory effects and mechanisms of NM-NPs without drug loading, and provided new insights and evidence for understanding their biological effects and safety, as well as developing more effective and safe targeted drug delivery systems.

Neutrophil Nanodecoys Inhibit Tumor Metastasis by Blocking the Interaction between Tumor Cells and Neutrophils.

Cancer metastasis is the main cause of cancer-related deaths and involves the interaction between tumor cells and neutrophils. In this study, we developed activated neutrophil membrane-coated nanoparticles (aNEM NPs) as nanodecoys to block neutrophil-mediated cancer metastasis. The aNEM NPs were fabricated by cloaking poly(lactic acid) nanoparticles with membranes derived from activated neutrophils and inherited the functional proteins of activated neutrophils. We demonstrated that aNEM NPs could interfere with the recruitment of neutrophils to the primary tumor and premetastatic niches, inhibit the adhesion of neutrophils to tumor vascular endothelium and circulating tumor cells (CTCs), and disrupt the formation of CTC-neutrophil clusters in vitro and in vivo. In 4T1-bearing mice, aNEM NPs could effectively reduce breast cancer metastasis to various organs in mice. Our results suggest that aNEM NPs are a promising nanomedicine for preventing or treating cancer metastasis by acting as neutrophil nanodecoys.

Overexpression of 18S rRNA methyltransferase CrBUD23 enhances biomass and lutein content in Chlamydomonas reinhardtii.

Post-transcriptional modification of nucleic acids including transfer RNA (tRNA), ribosomal RNA (rRNA) and messenger RNA (mRNA) is vital for fine-tunning of mRNA translation. Methylation is one of the most widespread post-transcriptional modifications in both eukaryotes and prokaryotes. HsWBSCR22 and ScBUD23 encodes a 18S rRNA methyltransferase that positively regulates cell growth by mediating ribosome maturation in human and yeast, respectively. However, presence and function of 18S rRNA methyltransferase in green algae are still elusive. Here, through bioinformatic analysis, we identified CrBUD23 as the human WBSCR22 homolog in genome of the green algae model organism Chlamydonomas reinhardtii. CrBUD23 was a conserved putative 18S rRNA methyltransferase widely exited in algae, plants, insects and mammalians. Transcription of CrBUD23 was upregulated by high light and down-regulated by low light, indicating its role in photosynthesis and energy metabolism. To characterize its biological function, coding sequence of CrBUD23 fused with a green fluorescence protein (GFP) tag was derived by 35S promoter and stably integrated into Chlamydomonas genome by glass bead-mediated transformation. Compared to C. reinhardtii wild type CC-5325, transgenic strains overexpressing CrBUD23 resulted in accelerated cell growth, thereby leading to elevated biomass, dry weight and protein content. Moreover, overexpression of CrBUD23 increased content of photosynthetic pigments but not elicit the activation of antioxidative enzymes, suggesting CrBUD23 favors growth and proliferation in the trade-off with stress responses. Bioinformatic analysis revealed the G1177 was the putative methylation site in 18S rRNA of C. reinhardtii CC-849. G1177 was conserved in other Chlamydonomas isolates, indicating the conserved methyltransferase activity of BUD23 proteins. In addition, CrTrm122, the homolog of BUD23 interactor Trm112, was found involved in responses to high light as same as CrBUD23. Taken together, our study revealed that cell growth, protein content and lutein accumulation of Chlamydomonas were positively regulated by the 18S rRNA methyltransferase CrBUD23, which could serve as a promising candidate for microalgae genetic engineering.

The aldolase inhibitor aldometanib mimics glucose starvation to activate lysosomal AMPK.

The activity of 5'-adenosine monophosphate-activated protein kinase (AMPK) is inversely correlated with the cellular availability of glucose. When glucose levels are low, the glycolytic enzyme aldolase is not bound to fructose-1,6-bisphosphate (FBP) and, instead, signals to activate lysosomal AMPK. Here, we show that blocking FBP binding to aldolase with the small molecule aldometanib selectively activates the lysosomal pool of AMPK and has beneficial metabolic effects in rodents. We identify aldometanib in a screen for aldolase inhibitors and show that it prevents FBP from binding to v-ATPase-associated aldolase and activates lysosomal AMPK, thereby mimicking a cellular state of glucose starvation. In male mice, aldometanib elicits an insulin-independent glucose-lowering effect, without causing hypoglycaemia. Aldometanib also alleviates fatty liver and nonalcoholic steatohepatitis in obese male rodents. Moreover, aldometanib extends lifespan and healthspan in both Caenorhabditis elegans and mice. Taken together, aldometanib mimics and adopts the lysosomal AMPK activation pathway associated with glucose starvation to exert physiological roles, and might have potential as a therapeutic for metabolic disorders in humans.

Low-dose metformin targets the lysosomal AMPK pathway through PEN2.

Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects1-4. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action4,5; however, the direct molecular target of metformin remains unknown. Here we show that clinically relevant concentrations of metformin inhibit the lysosomal proton pump v-ATPase, which is a central node for AMPK activation following glucose starvation6. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase7, as a binding partner of metformin with a dissociation constant at micromolar levels. Metformin-bound PEN2 forms a complex with ATP6AP1, a subunit of the v-ATPase8, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of PEN2 or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects. Furthermore, knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan. Together, these findings reveal that metformin binds PEN2 and initiates a signalling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects.

Transient Receptor Potential V Channels Are Essential for Glucose Sensing by Aldolase and AMPK.

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.

Hierarchical activation of compartmentalized pools of AMPK depends on severity of nutrient or energy stress.

AMPK, a master regulator of metabolic homeostasis, is activated by both AMP-dependent and AMP-independent mechanisms. The conditions under which these different mechanisms operate, and their biological implications are unclear. Here, we show that, depending on the degree of elevation of cellular AMP, distinct compartmentalized pools of AMPK are activated, phosphorylating different sets of targets. Low glucose activates AMPK exclusively through the AMP-independent, AXIN-based pathway in lysosomes to phosphorylate targets such as ACC1 and SREBP1c, exerting early anti-anabolic and pro-catabolic roles. Moderate increases in AMP expand this to activate cytosolic AMPK also in an AXIN-dependent manner. In contrast, high concentrations of AMP, arising from severe nutrient stress, activate all pools of AMPK independently of AXIN. Surprisingly, mitochondrion-localized AMPK is activated to phosphorylate ACC2 and mitochondrial fission factor (MFF) only during severe nutrient stress. Our findings reveal a spatiotemporal basis for hierarchical activation of different pools of AMPK during differing degrees of stress severity.

Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK.

The major energy source for most cells is glucose, from which ATP is generated via glycolysis and/or oxidative metabolism. Glucose deprivation activates AMP-activated protein kinase (AMPK), but it is unclear whether this activation occurs solely via changes in AMP or ADP, the classical activators of AMPK. Here, we describe an AMP/ADP-independent mechanism that triggers AMPK activation by sensing the absence of fructose-1,6-bisphosphate (FBP), with AMPK being progressively activated as extracellular glucose and intracellular FBP decrease. When unoccupied by FBP, aldolases promote the formation of a lysosomal complex containing at least v-ATPase, ragulator, axin, liver kinase B1 (LKB1) and AMPK, which has previously been shown to be required for AMPK activation. Knockdown of aldolases activates AMPK even in cells with abundant glucose, whereas the catalysis-defective D34S aldolase mutant, which still binds FBP, blocks AMPK activation. Cell-free reconstitution assays show that addition of FBP disrupts the association of axin and LKB1 with v-ATPase and ragulator. Importantly, in some cell types AMP/ATP and ADP/ATP ratios remain unchanged during acute glucose starvation, and intact AMP-binding sites on AMPK are not required for AMPK activation. These results establish that aldolase, as well as being a glycolytic enzyme, is a sensor of glucose availability that regulates AMPK.