JMJD5, a Jumonji C domain-containing dioxygenase, is important for embryonic development and cancer growth. Here, we show that JMJD5 is up-regulated by hypoxia and is crucial for hypoxiainduced cell proliferation. JMJD5 interacts directly with pyruvate kinase muscle isozyme (PKM)2 to modulate metabolic flux in cancer cells. The JMJD5-PKM2 interaction resides at the intersubunit interface region of PKM2, which hinders PKM2 tetramerization and blocks pyruvate kinase activity. This interaction also influences translocation of PKM2 into the nucleus and promotes hypoxiainducible factor (HIF)-1α-mediated transactivation. JMJD5 knockdown inhibits the transcription of the PKM2-HIF-1α target genes involved in glucose metabolism, resulting in a reduction of glucose uptake and lactate secretion in cancer cells. JMJD5, along with PKM2 and HIF-1α, is recruited to the hypoxia response element site in the lactate dehydrogenase A and PKM2 loci and mediates the recruitment of the latter two proteins. Our data uncover a mechanism whereby PKM2 can be regulated by factor-bindinginduced homo/heterooligomeric restructuring, paving the way to cell metabolic reprogram.Warburg effect | aerobic glycolysis | breast cancer | cancer metabolism J MJD5 is a Jumonji C domain-containing dioxygenase shown to be involved in lysine demethylation (1-3) and hydroxylation functions (4). Although the exact cellular substrates and functions of JMJD5 remain unclear, JMJD5 was shown to positively regulate cyclin A1 but negatively regulate p53 and p21 (1-3). Knockdown of JMJD5 in Michigan Cancer Foundation (MCF)-7 cells inhibits cell proliferation (1), and JMJD5 −/− embryos showed severe growth retardation, resulting in embryonic lethality at the midgestation stage (3). These data, together with its general overexpression in tumor tissues, implicate a role of JMJD5 in carcinogenesis. In this paper, we define a role of JMJD5 in regulating tumor metabolism under normoxic and hypoxic conditions through its interaction with pyruvate kinase muscle isozyme (PKM)2.One of the hallmarks of cancer cells is their altered metabolism, referred to as aerobic glycolysis, or the Warburg effect (5). This generally involves an increased uptake of glucose, use of intracellular glucose to pyruvate via glycolysis, and the conversion into lactate in the presence of sufficient oxygen. Along this metabolic flux, PKM1 or its spliced variant, PKM2, which dephosphorylates phosphoenolpyruvate (PEP) into pyruvate, the last step of glycolysis, is an important signal integrator whose activities determine the cytosolic level of pyruvate, thereby affecting subsequent metabolic flow to lactate, tricarboxylic acid cycle or biosynthetic pathway (6). Enzymatically, PKM2, an embryonic isoform found abundantly in tumor cells, is less active than PKM1, which allows the accumulation of glycolytic intermediates and diversion into biosynthetic pathways, demanded by rapid-proliferating cells.As a pivotal regulator of tumor metabolism, PKM2's activity is further modulated by allosteric regulation vi...

Background: Stroke is a leading cause of adult disability that can severely compromise patients' quality of life, yet no effective medication currently exists to accelerate rehabilitation. A variety of circular RNA (circRNAs) molecules are known to function in ischemic brain injury. Lentivirus-based expression systems have been widely used in basic studies of circRNAs, but safety issues with such delivery systems have limited exploration of potential therapeutic roles for circRNAs. Methods: Circular RNA SCMH1 (circSCMH1) was screened from the plasma of acute ischemic stroke (AIS) patients using circRNA microarrays. Engineered RVG-circSCMH1-extracellular vesicles (RVG-circSCMH1-EVs) were generated to selectively deliver circSCMH1 to the brain. Nissl staining was used to examine infarct size. Behavioral tasks were performed to evaluate motor functions in both rodent and nonhuman primate ischemic stroke models. Golgi staining and immunostaining were used to examine neuroplasticity and glial activation. Proteomic assays and RNA-seq data combined with transcriptional profiling were used to identify downstream targets of circSCMH1. Results: CircSCMH1 levels were significantly decreased in plasma of AIS patients, offering significant power in predicting stroke outcomes. The decreased levels of circSCMH1 were further confirmed in the plasma and peri-infarct cortex of photothrombotic (PT) stroke mice. Beyond demonstrating proof-of-concept for an RNA drug delivery technology, we observed that circSCMH1 treatment improved functional recovery post stroke in both mice and monkeys, and discovered that circSCMH1 enhanced the neuronal plasticity and also inhibited glial activation and peripheral immune cell infiltration. Mechanistically, circSCMH1 binds to the transcription factor MeCP2, thereby releasing repression of MeCP2 target gene transcription. Conclusions: RVG-circSCMH1-EVs afford protection by promoting functional recovery in the rodent and the nonhuman primate ischemic stroke models. Our study presents a potentially widely applicable nucleotide drug delivery technology and demonstrates the basic mechanism of how circRNAs can be therapeutically exploited to improve post-stroke outcomes.

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Prelabor rupture of the fetal membranes affects approximately 10% of women at term, resulting in an increased risk of maternal and neonatal infection. Evidence suggests that membrane rupture is related to biochemical processes involving the extracellular matrix of the membranes. We tested the hypothesis that prelabor ruptured membranes are characterized by reduced collagen concentrations, altered collagen cross-link profiles, and increased concentrations of biomarkers of oxidative damage. We also set out to determine whether these effects are modulated by ascorbic acid status. In a case-control study, we explored the role that ascorbic acid, oxidative stress, collagen, and collagen cross-links play in determining membrane integrity and developed a functional assay to assess membrane proteolytic susceptibility. Prelabor ruptured membrane had a reduced ascorbic acid concentration in comparison with controls while protein carbonyl and malondialdehyde concentrations were increased. Collagen concentrations were also reduced in prelabor ruptured membrane, and while the concentration of collagen cross-links was not significantly different between prelabor and timely ruptured membrane, there was a regional variation in cross-link ratio within the amniotic sac. Proteolytic resistance in vitro was reduced in prelabor ruptured membrane and also exhibited regional variation within the amniotic sac. Our findings are strongly supportive of a role for the enhanced degradation of membrane collagen in the determination of prelabor rupture of fetal membranes. The formation of the rupture initiation site is a function of a regional variation in collagen cross-link ratio. Tissue ascorbic acid status may be an important mediator of these processes.

Ca2+ plays a significant role in linking the induction of apoptosis. The key anti-apoptotic protein, Bcl-2, has been reported to regulate the movement of Ca2+ across the ER membrane, but the exact effect of Bcl-2 on Ca2+ levels remains controversial. Store-operated Ca2+ entry (SOCE), a major mode of Ca2+ uptake in non-excitable cells, is activated by depletion of Ca2+ in the ER. Depletion of Ca2+ in the ER causes translocation of the SOC channel activator, STIM1, to the plasma membrane. Thereafter, STIM1 binds to Orai1 or/and TRPC1 channels, forcing them to open and thereby allow Ca2+ entry. In addition, several anti-cancer drugs have been reported to induce apoptosis of cancer cells via the SOCE pathway. However, the detailed mechanism underlying the regulation of SOCE by Bcl-2 is not well understood. In this study, a three-amino acid mutation within the Bcl-2 BH1 domain was generated to verify the role of Bcl-2 in Ca2+ handling during ER stress. The subcellular localization of the Bcl-2 mutant (mt) is similar to that in the wild-type Bcl-2 (WT) in the ER and mitochondria. We found that mt enhanced thapsigargin and tunicamycin-induced apoptosis through ER stress-mediated apoptosis but not through the death receptor- and mitochondria-dependent apoptosis, while WT prevented thapsigargin- and tunicamycin-induced apoptosis. In addition, mt depleted Ca2+ in the ER lumen and also increased the expression of SOCE-related molecules. Therefore, a massive Ca2+ influx via SOCE contributed to caspase activation and apoptosis. Furthermore, inhibiting SOCE or chelating either extracellular or intracellular Ca2+ inhibited mt-mediated apoptosis. In brief, our results explored the critical role of Bcl-2 in Ca2+ homeostasis and the modulation of ER stress.