Non-Essential Amino Acid Availability Influences Proteostasis and Breast Cancer Cell Survival During Proteotoxic Stress.

Protein homeostasis (proteostasis) regulates tumor growth and proliferation when cells are exposed to proteotoxic stress, such as during treatment with certain chemotherapeutics. Consequently, cancer cells depend to a greater extent on stress signaling, and require the integrated stress response (ISR), amino acid metabolism, and efficient protein folding and degradation pathways to survive. To define how these interconnected pathways are wired when cancer cells are challenged with proteotoxic stress, we investigated how amino acid abundance influences cell survival when Hsp70, a master proteostasis regulator, is inhibited. We previously demonstrated that cancer cells exposed to a specific Hsp70 inhibitor induce the ISR via the action of two sensors, GCN2 and PERK, in stress-resistant and sensitive cells, respectively. In resistant cells, the induction of GCN2 and autophagy supported resistant cell survival, yet the mechanism by which these events were induced remained unclear. We now report that amino acid availability reconfigures the proteostasis network. Amino acid supplementation, and in particular arginine addition, triggered cancer cell death by blocking autophagy. Consistent with the importance of amino acid availability, which when limited activates GCN2, resistant cancer cells succumbed when challenged with a potentiator for another amino acid sensor, mTORC1, in conjunction with Hsp70 inhibition. IMPLICATIONS: These data position amino acid abundance, GCN2, mTORC1, and autophagy as integrated therapeutic targets whose coordinated modulation regulates the survival of proteotoxic-resistant breast cancer cells.

Discovery of bactericides as an acute mitochondrial membrane damage inducer.

Mitochondria evolved from endosymbiotic bacteria to become essential organelles of eukaryotic cells. The unique lipid composition and structure of mitochondrial membranes are critical for the proper functioning of mitochondria. However, stress responses that help maintain the mitochondrial membrane integrity are not well understood. One reason for this lack of insight is the absence of efficient tools to specifically damage mitochondrial membranes. Here, through a compound screen, we found that two bis-biguanide compounds, chlorhexidine and alexidine, modified the activity of the inner mitochondrial membrane (IMM)-resident protease OMA1 by altering the integrity of the IMM. These compounds are well-known bactericides whose mechanism of action has centered on their damage-inducing activity on bacterial membranes. We found alexidine binds to the IMM likely through the electrostatic interaction driven by the membrane potential as well as an affinity for anionic phospholipids. Electron microscopic analysis revealed that alexidine severely perturbated the cristae structure. Notably, alexidine evoked a specific transcriptional/proteostasis signature that was not induced by other typical mitochondrial stressors, highlighting the unique property of alexidine as a novel mitochondrial membrane stressor. Our findings provide a chemical-biological tool that should enable the delineation of mitochondrial stress-signaling pathways required to maintain the mitochondrial membrane homeostasis.

Nitroalkene fatty acids modulate bile acid metabolism and lung function in obese asthma.

Bile acid profiles are altered in obese individuals with asthma. Thus, we sought to better understand how obesity-related systemic changes contribute to lung pathophysiology. We also test the therapeutic potential of nitro-oleic acid (NO2-OA), a regulator of metabolic and inflammatory signaling pathways, to mitigate allergen and obesity-induced lung function decline in a murine model of asthma. Bile acids were measured in the plasma of healthy subjects and individuals with asthma and serum and lung tissue of mice with and without allergic airway disease (AAD). Lung function, indices of inflammation and hepatic bile acid enzyme expression were measured in obese mice with house dust mite-induced AAD treated with vehicle or NO2-OA. Serum levels of glycocholic acid and glycoursodeoxycholic acid clinically correlate with body mass index and airway hyperreactivity whereas murine levels of ß-muricholic acid and tauro-ß-muricholic acid were significantly increased and positively correlated with impaired lung function in obese mice with AAD. NO2-OA reduced murine bile acid levels by modulating hepatic expression of bile acid synthesis enzymes, with a concomitant reduction in small airway resistance and tissue elastance. Bile acids correlate to body mass index and lung function decline and the signaling actions of nitroalkenes can limit AAD by modulating bile acid metabolism, revealing a potential pharmacologic approach to improving the current standard of care.

Sustained Dysbiosis and Decreased Fecal Short-Chain Fatty Acids after Traumatic Brain Injury and Impact on Neurologic Outcome.

Traumatic brain injury (TBI) alters microbial populations present in the gut, which may impact healing and tissue recovery. However, the duration and impact of these changes on outcome from TBI are unknown. Short-chain fatty acids (SCFAs), produced by bacterial fermentation of dietary fiber, are important signaling molecules in the microbiota gut-brain axis. We hypothesized that TBI would lead to a sustained reduction in SCFA producing bacteria, fecal SCFAs concentration, and administration of soluble SCFAs would improve functional outcome after TBI. Adult mice (n = 10) had the controlled cortical impact (CCI) model of TBI performed (6 m/sec, 2-mm depth, 50-msec dwell). Stool samples were collected serially until 28 days after CCI and analyzed for SCFA concentration by high-performance liquid chromatography-mass spectrometry/mass spectrometry and microbiome analyzed by 16S gene sequencing. In a separate experiment, mice (n = 10/group) were randomized 2 weeks before CCI to standard drinking water or water supplemented with the SCFAs acetate (67.5 mM), propionate (25.9 mM), and butyrate (40 mM). Morris water maze performance was assessed on post-injury Days 14-19. Alpha diversity remained stable until 72 h, at which point a decline in diversity was observed without recovery out to 28 days. The taxonomic composition of post-TBI fecal samples demonstrated depletion of bacteria from Lachnospiraceae, Ruminococcaceae, and Bacteroidaceae families, and enrichment of bacteria from the Verrucomicrobiaceae family. Analysis from paired fecal samples revealed a reduction in total SCFAs at 24 h and 28 days after TBI. Acetate, the most abundant SCFA detected in the fecal samples, was reduced at 7 days and 28 days after TBI. SCFA administration improved spatial learning after TBI versus standard drinking water. In conclusion, TBI is associated with reduced richness and diversity of commensal microbiota in the gut and a reduction in SCFAs detected in stool. Supplementation of soluble SCFAs improves spatial learning after TBI.

BOLA (BolA Family Member 3) Deficiency Controls Endothelial Metabolism and Glycine Homeostasis in Pulmonary Hypertension.

BACKGROUND: Deficiencies of iron-sulfur (Fe-S) clusters, metal complexes that control redox state and mitochondrial metabolism, have been linked to pulmonary hypertension (PH), a deadly vascular disease with poorly defined molecular origins. BOLA3 (BolA Family Member 3) regulates Fe-S biogenesis, and mutations in BOLA3 result in multiple mitochondrial dysfunction syndrome, a fatal disorder associated with PH. The mechanistic role of BOLA3 in PH remains undefined. METHODS: In vitro assessment of BOLA3 regulation and gain- and loss-of-function assays were performed in human pulmonary artery endothelial cells using siRNA and lentiviral vectors expressing the mitochondrial isoform of BOLA3. Polymeric nanoparticle 7C1 was used for lung endothelium-specific delivery of BOLA3 siRNA oligonucleotides in mice. Overexpression of pulmonary vascular BOLA3 was performed by orotracheal transgene delivery of adeno-associated virus in mouse models of PH. RESULTS: In cultured hypoxic pulmonary artery endothelial cells, lung from human patients with Group 1 and 3 PH, and multiple rodent models of PH, endothelial BOLA3 expression was downregulated, which involved hypoxia inducible factor-2α-dependent transcriptional repression via histone deacetylase 1-mediated histone deacetylation. In vitro gain- and loss-of-function studies demonstrated that BOLA3 regulated Fe-S integrity, thus modulating lipoate-containing 2-oxoacid dehydrogenases with consequent control over glycolysis and mitochondrial respiration. In contexts of siRNA knockdown and naturally occurring human genetic mutation, cellular BOLA3 deficiency downregulated the glycine cleavage system protein H, thus bolstering intracellular glycine content. In the setting of these alterations of oxidative metabolism and glycine levels, BOLA3 deficiency increased endothelial proliferation, survival, and vasoconstriction while decreasing angiogenic potential. In vivo, pharmacological knockdown of endothelial BOLA3 and targeted overexpression of BOLA3 in mice demonstrated that BOLA3 deficiency promotes histological and hemodynamic manifestations of PH. Notably, the therapeutic effects of BOLA3 expression were reversed by exogenous glycine supplementation. CONCLUSIONS: BOLA3 acts as a crucial lynchpin connecting Fe-S-dependent oxidative respiration and glycine homeostasis with endothelial metabolic reprogramming critical to PH pathogenesis. These results provide a molecular explanation for the clinical associations linking PH with hyperglycinemic syndromes and mitochondrial disorders. These findings also identify novel metabolic targets, including those involved in epigenetics, Fe-S biogenesis, and glycine biology, for diagnostic and therapeutic development.

CMPF, a Metabolite Formed Upon Prescription Omega-3-Acid Ethyl Ester Supplementation, Prevents and Reverses Steatosis.

Prescription ω-3 fatty acid ethyl ester supplements are commonly used for the treatment of hypertriglyceridemia. However, the metabolic profile and effect of the metabolites formed by these treatments remain unknown. Here we utilized unbiased metabolomics to identify 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) as a significant metabolite of the ω-3-acid ethyl ester prescription Lovaza™ in humans. Administration of CMPF to mice before or after high-fat diet feeding at exposures equivalent to those observed in humans increased whole-body lipid metabolism, improved insulin sensitivity, increased beta-oxidation, reduced lipogenic gene expression, and ameliorated steatosis. Mechanistically, we find that CMPF acutely inhibits ACC activity, and induces long-term loss of SREBP1c and ACC1/2 expression. This corresponds to an induction of FGF21, which is required for long-term steatosis protection, as FGF21KO mice are refractory to the improved metabolic effects. Thus, CMPF treatment in mice parallels the effects of human Lovaza™ supplementation, revealing that CMPF may contribute to the improved metabolic effects observed with ω-3 fatty acid prescriptions.