Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic resolution structures for well-ordered protein complexes with sizes ≥ ∼200 kDa. ...Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of the cancer target isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: (1) crossing 2 Å resolution and (2) obtaining structures of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.
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•Near-atomic-resolution structure of isocitrate dehydrogenase, a < 100 kDa enzyme•1.8 Å resolution cryo-EM map of 334 kDa glutamate dehydrogenase•2.8 Å resolution cryo-EM map of lactate dehydrogenase, a 145 kDa protein•Localization of small-molecule inhibitors bound to metabolic protein complexes
By using cryo-EM methods, the structure of small metabolic enzymes as well as the localization of small-molecule inhibitors that bind to them can be determined at near-atomic resolution.
Control of intracellular reactive oxygen species (ROS) concentrations is critical for cancer cell survival. We show that, in human lung cancer cells, acute increases in intracellular concentrations ...of ROS caused inhibition of the glycolytic enzyme pyruvate kinase M2 (PKM2) through oxidation of Cys³⁵⁸. This inhibition of PKM2 is required to divert glucose flux into the pentose phosphate pathway and thereby generate sufficient reducing potential for detoxification of ROS. Lung cancer cells in which endogenous PKM2 was replaced with the Cys³⁵⁸ to Ser³⁵⁸ oxidation-resistant mutant exhibited increased sensitivity to oxidative stress and impaired tumor formation in a xenograft model. Besides promoting metabolic changes required for proliferation, the regulatory properties of PKM2 may confer an additional advantage to cancer cells by allowing them to withstand oxidative stress.
Loss of function mutations in Kelch-like ECH Associated Protein 1 (KEAP1), or gain-of-function mutations in nuclear factor erythroid 2-related factor 2 (NRF2), are common in non-small cell lung ...cancer (NSCLC) and associated with therapeutic resistance. To discover novel NRF2 inhibitors for targeted therapy, we conducted a quantitative high-throughput screen using a diverse set of ∼400 000 small molecules (Molecular Libraries Small Molecule Repository Library, MLSMR) at the National Center for Advancing Translational Sciences. We identified ML385 as a probe molecule that binds to NRF2 and inhibits its downstream target gene expression. Specifically, ML385 binds to Neh1, the Cap ‘N’ Collar Basic Leucine Zipper (CNC-bZIP) domain of NRF2, and interferes with the binding of the V-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homologue G (MAFG)-NRF2 protein complex to regulatory DNA binding sequences. In clonogenic assays, when used in combination with platinum-based drugs, doxorubicin or taxol, ML385 substantially enhances cytotoxicity in NSCLC cells, as compared to single agents. ML385 shows specificity and selectivity for NSCLC cells with KEAP1 mutation, leading to gain of NRF2 function. In preclinical models of NSCLC with gain of NRF2 function, ML385 in combination with carboplatin showed significant antitumor activity. We demonstrate the discovery and validation of ML385 as a novel and specific NRF2 inhibitor and conclude that targeting NRF2 may represent a promising strategy for the treatment of advanced NSCLC.
In this study, we examine whether youth who are exposed to more weapons violence are subsequently more likely to behave violently with weapons. We use data collected with a 3‐cohort, 4‐wave, 10‐year ...longitudinal study of 426 high‐risk youth from Flint, Michigan, who were second, fourth, or ninth‐graders in 2006–2007. The data were obtained from individual interviews with the youth, their parents, and their teachers, from archival school and criminal justice records, and from geo‐coded criminal offense data. These data show that early exposure to weapons violence significantly correlates at modest levels with weapon carrying, weapon use or threats‐to‐use, arrests for weapons use, and criminally violent acts 10 years later. Multiple regression analyses, controlling for children's initial aggressiveness, intellectual achievement, and parents' income, education, and aggression, reveal statistically significant independent 10‐year effects: (1) more early exposure to weapon use within the family predicts more using or threatening to use a gun; (2) more cumulative early violent video game playing predicts more gun using or threatening to use weapons, and normative beliefs that gun use is acceptable; (3) more cumulative early exposure to neighborhood gun violence predicts more arrests for a weapons crime; and (4) more cumulative early exposure to movie violence predicts more weapon carrying. We argue that youth who observe violence with weapons, whether in the family, among peers, or through the media or video games, are likely to be infected from exposure with a social–cognitive–emotional disease that increases their own risk of behaving violently with weapons later in life.
The tumour microenvironment contributes to cancer metastasis and drug resistance. However, most high throughput screening (HTS) assays for drug discovery use cancer cells grown in monolayers. Here we ...show that a multilayered culture containing primary human fibroblasts, mesothelial cells and extracellular matrix can be adapted into a reliable 384- and 1,536-multi-well HTS assay that reproduces the human ovarian cancer (OvCa) metastatic microenvironment. We validate the identified inhibitors in secondary in vitro and in vivo biological assays using three OvCa cell lines: HeyA8, SKOV3ip1 and Tyk-nu. The active compounds directly inhibit at least two of the three OvCa functions: adhesion, invasion and growth. In vivo, these compounds prevent OvCa adhesion, invasion and metastasis, and improve survival in mouse models. Collectively, these data indicate that a complex three-dimensional culture of the tumour microenvironment can be adapted for quantitative HTS and may improve the disease relevance of assays used for drug screening.
Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid, and ...lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation.
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•PEPCK promotes tumor growth by increasing glucose and glutamine metabolism•PEPCK increases anabolic metabolism, including ribose synthesis from glutamine•PEPCK promotes mTORC1 activity•PEPCK increases glycolysis and proliferation via mTORC1 activation
Montal et al. report that PEPCK increases tumor growth by promoting metabolic flexibility. PEPCK increases the utilization of both glucose and glutamine toward anabolic metabolism. In addition, the ability of PEPCK to increase glucose metabolism and cell proliferation was mediated in part via activation of mTORC1.
Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to ...interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC50 of 1.1 μm in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines. ML364, and not the inactive analog 2, was antiproliferative in cancer cell lines. Consistent with the role of cyclin D1 in DNA damage response, ML364 also caused a decrease in homologous recombination-mediated DNA repair. These effects by a small molecule inhibitor support a key role for USP2 as a regulator of cell cycle, DNA repair, and tumor cell growth.
This WHO-commissioned review contributed to the update of complementary feeding recommendations, synthesizing evidence on effects of unhealthy food and beverage consumption in children on overweight ...and obesity. We searched PubMed (Medline), Cochrane CENTRAL, and Embase for articles, irrespective of language or geography. Inclusion criteria were: 1) randomized controlled trials (RCTs), non-RCTs, cohort studies, and pre/post studies with control; 2) participants aged ≤10.9 y at exposure; 3) studies reporting greater consumption of unhealthy foods/beverages compared with no or low consumption; 4) studies assessing anthropometric and/or body composition; and 5) publication date ≥1971. Unhealthy foods and beverages were defined using nutrient- and food-based approaches. Risk of bias was assessed using the ROBINS-I (risk of bias in nonrandomized studies of interventions version I) and RoB2 Cochrane RoB (version 2) tools for nonrandomized and randomized studies, respectively. Narrative synthesis was complemented by meta-analyses where appropriate. Certainty of evidence was assessed using Grading of Recommendations Assessment, Development, and Evaluation. Of 26,542 identified citations, 60 studies from 71 articles were included. Most studies were observational (59/60), and no included studies were from low-income countries. The evidence base was low quality, as assessed by ROBINS-I and RoB2 tools. Evidence synthesis was limited by the different interventions and comparators across studies. Evidence indicated that consumption of sugar-sweetened beverages (SSBs) and unhealthy foods in childhood may increase BMI/BMI z-score, percentage body fat, or odds of overweight/obesity (low certainty of evidence). Artificially sweetened beverages and 100% fruit juice consumption make little/no difference to BMI, percentage body fat, or overweight/obesity outcomes (low certainty of evidence). Meta-analyses of a subset of studies indicated a positive association between SSB intake and percentage body fat, but no association with change in BMI and BMI z-score. High-quality epidemiological studies that are designed to assess the effects of unhealthy food consumption during childhood on risk of overweight/obesity are needed to contribute to a more robust evidence base upon which to design policy recommendations.
This protocol was registered at https://www.crd.york.ac.uk/PROSPERO as CRD42020218109.
Statement of Significance: This systematic review was the first to synthesize evidence on the effects of consuming unhealthy foods and beverages in children aged ≤10.9 y across low-, middle-, and high-income country settings. This review identified a major evidence gap from low- and middle- income countries. Consumption of sugar-sweetened beverages and unhealthy foods in childhood may increase BMI, percentage body fat, or odds of overweight/obesity, whereas consumption of artificially sweetened beverages and 100% fruit juice has less demonstrable effects on these outcomes.
Extrapituitary prolactin (Prl) is produced in humans and rodents; however, little is known about its in vivo regulation or physiological function. We now report that autocrine prolactin is required ...for terminal mammary epithelial differentiation during pregnancy and that its production is regulated by the Pten-PI3K-Akt pathway. Conditional activation of the PI3K-Akt pathway in the mammary glands of virgin mice by either Akt1 expression or Pten deletion rapidly induced terminal mammary epithelial differentiation accompanied by the synthesis of milk despite the absence of lobuloalveolar development. Surprisingly, we found that mammary differentiation was due to the PI3K-Akt-dependent synthesis and secretion of autocrine prolactin and downstream activation of the prolactin receptor (Prlr)-Jak-Stat5 pathway. Consistent with this, Akt-induced mammary differentiation was abrogated in Prl(-/-), Prlr(-/-), and Stat5(-/-) mice. Furthermore, cells treated with conditioned medium from mammary glands in which Akt had been activated underwent rapid Stat5 phosphorylation in a manner that was blocked by inhibition of Jak2, treatment with an anti-Prl antibody, or deletion of the prolactin gene. Demonstrating a physiological requirement for autocrine prolactin, mammary glands from lactation-defective Akt1(-/-);Akt2(+/-) mice failed to express autocrine prolactin or activate Stat5 during late pregnancy despite normal levels of circulating serum prolactin and pituitary prolactin production. Our findings reveal that PI3K-Akt pathway activation is necessary and sufficient to induce autocrine prolactin production in the mammary gland, Stat5 activation, and terminal mammary epithelial differentiation, even in the absence of the normal developmental program that prepares the mammary gland for lactation. Together, these findings identify a function for autocrine prolactin during normal development and demonstrate its endogenous regulation by the PI3K-Akt pathway.
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