Intrinsic Mechanisms Underlying Hypoxia-Tolerant Mitochondrial Phenotype During Hypoxia-Reoxygenation Stress in a Marine Facultative Anaerobe, the Blue Mussel Mytilus edulis

Hypoxia is common in marine environments and a major stressor for marine organisms inhabiting benthic and intertidal zones. Several studies have explored the responses of these organisms to hypoxic stress at the whole organism level with a focus on energy metabolism and mitochondrial response, but the instrinsic mitochondrial responses that support the organelle’s function under hypoxia and reoxygenation (H/R) stress are not well understood. We studied the effects of acute H/R stress (10 min anoxia followed by 15 min reoxygenation) on mitochondrial respiration, production of reactive oxygen species (ROS) and posttranslational modifications (PTM) of the proteome in a marine facultative anaerobe, the blue mussel Mytilus edulis. The mussels’ mitochondria showed increased OXPHOS respiration and suppressed proton leak resulting in a higher coupling efficiency after H/R stress. ROS production decreased in both the resting (LEAK) and phosphorylating (OXPHOS) state indicating that M. edulis was able to prevent oxidative stress and mitochondrial damage during reoxygenation. Hypoxia did not lead to rearrangement of the mitochondrial supercomplexes but impacted the mitochondrial phosphoproteome including the proteins involved in OXPHOS, amino acid- and fatty acid catabolism, and protein quality control. This study indicates that mussels’ mitochondria possess intrinsic mechanisms (including regulation via reversible protein phosphorylation) that ensure high respiratory flux and mitigate oxidative damage during H/R stress and contribute to the hypoxia-tolerant mitochondrial phenotype of this metabolically plastic species.

Metabolomics provide new insights into mechanisms of Wolbachia-induced paternal defects in Drosophila melanogaster

Wolbachia is a group of intracellular symbiotic bacteria that widely infect arthropods and nematodes. Wolbachia infection can regulate host reproduction with the most common phenotype in insects being cytoplasmic incompatibility (CI), which results in embryonic lethality when uninfected eggs fertilized with sperms from infected males. This suggests that CI-induced defects are mainly in paternal side. However, whether Wolbachia-induced metabolic changes play a role in the mechanism of paternal-linked defects in embryonic development is not known. In the current study, we first use untargeted metabolomics method with LC-MS to explore how Wolbachia infection influences the metabolite profiling of the insect hosts. The untargeted metabolomics revealed 414 potential differential metabolites between Wolbachia-infected and uninfected 1-day-old (1d) male flies. Most of the differential metabolites were significantly up-regulated due to Wolbachia infection. Thirty-four metabolic pathways such as carbohydrate, lipid and amino acid, and vitamin and cofactor metabolism were affected by Wolbachia infection. Then, we applied targeted metabolomics analysis with GC-MS and showed that Wolbachia infection resulted in an increased energy expenditure of the host by regulating glycometabolism and fatty acid catabolism, which was compensated by increased food uptake. Furthermore, overexpressing two acyl-CoA catabolism related genes, Dbi (coding for diazepam-binding inhibitor) or Mcad (coding for medium-chain acyl-CoA dehydrogenase), ubiquitously or specially in testes caused significantly decreased paternal-effect egg hatch rate. Oxidative stress and abnormal mitochondria induced by Wolbachia infection disrupted the formation of sperm nebenkern. These findings provide new insights into mechanisms of Wolbachia-induced paternal defects from metabolic phenotypes.

Elucidating the genome of Emydura subglobosa through manual annotation and computational biology tools

Rapid advancements in automated genomic technologies have uncovered many unique findings about the turtle genome and its associated features including olfactory gene expansions and duplications of toll-like receptors. However, automated technologies often result in a high frequency of errors through the process of assembly and annotation and highlight the need for manual annotation. In this study, we have manually annotated four genes of the red-bellied short-neck turtle (Emydura subglobosa), an understudied outgroup of turtles representing a diverse lineage. We improved upon initial ab initio gene predictions through homology-based evidence and generated refined consensus models. Through functional, localization, and structural analyses of the predicted proteins, we have discovered conserved genes encoding proteins that play a role in C21-steroid hormone biosynthetic processes, Vitamin A uptake, collagen/elastin integrity, tumor suppression, and fatty acid catabolism. Overall, these findings further our knowledge about the genetic features underlying turtle physiology, morphology, and longevity, which could have important implications for the treatment of human diseases and evolutionary studies.

Mycobacterial fatty acid catabolism is repressed by FdmR to sustain lipogenesis and virulence

Host-derived fatty acids are an important carbon source for pathogenic mycobacteria during infection. How mycobacterial cells regulate the catabolism of fatty acids to serve the pathogenicity, however, remains unknown. Here, we identified a TetR-family transcriptional factor, FdmR, as the key regulator of fatty acid catabolism in the pathogen Mycobacterium marinum by combining use of transcriptomics, chromatin immunoprecipitation followed by sequencing, dynamic 13C-based flux analysis, metabolomics, and lipidomics. An M. marinum mutant deficient in FdmR was severely attenuated in zebrafish larvae and adult zebrafish. The mutant showed defective growth but high substrate consumption on fatty acids. FdmR was identified as a long-chain acyl-coenzyme A (acyl-CoA)–responsive repressor of genes involved in fatty acid degradation and modification. We demonstrated that FdmR functions as a valve to direct the flux of exogenously derived fatty acids away from β-oxidation toward lipid biosynthesis, thereby avoiding the overactive catabolism and accumulation of biologically toxic intermediates. Moreover, we found that FdmR suppresses degradation of long-chain acyl-CoAs endogenously synthesized through the type I fatty acid synthase. By modulating the supply of long-chain acyl-CoAs for lipogenesis, FdmR controls the abundance and chain length of virulence-associated lipids and mycolates and plays an important role in the impermeability of the cell envelope. These results reveal that despite the fact that host-derived fatty acids are used as an important carbon source, overactive catabolism of fatty acids is detrimental to mycobacterial cell growth and pathogenicity. This study thus presents FdmR as a potentially attractive target for chemotherapy.

Peroxisomes form intralumenal vesicles with roles in fatty acid catabolism and protein compartmentalization in Arabidopsis

Peroxisomes are vital organelles that compartmentalize critical metabolic reactions, such as the breakdown of fats, in eukaryotic cells. Although peroxisomes typically are considered to consist of a single membrane enclosing a protein lumen, more complex peroxisomal membrane structure has occasionally been observed in yeast, mammals, and plants. However, technical challenges have limited the recognition and understanding of this complexity. Here we exploit the unusually large size of Arabidopsis peroxisomes to demonstrate that peroxisomes have extensive internal membranes. These internal vesicles accumulate over time, use ESCRT (endosomal sorting complexes required for transport) machinery for formation, and appear to derive from the outer peroxisomal membrane. Moreover, these vesicles can harbor distinct proteins and do not form normally when fatty acid β-oxidation, a core function of peroxisomes, is impaired. Our findings suggest a mechanism for lipid mobilization that circumvents challenges in processing insoluble metabolites. This revision of the classical view of peroxisomes as single-membrane organelles has implications for all aspects of peroxisome biogenesis and function and may help address fundamental questions in peroxisome evolution.

93 Targeting sirt2 rescues the metabolic fitness and effector functions of tumor-reactive T cells within the metabolically restricted tumor microenvironment

BackgroundThe majority of cancer patients remain refractory to existing cancer immunotherapies. Despite the growing evidence that dysregulated metabolism contributes to the exhaustion of tumor-infiltrating T lymphocytes (TILs) and the loss of their effector functions within the metabolically restricted tumor microenvironment (TME), actionable targets to rescue metabolic fitness and anti-tumor activity of TILs remain elusive.Memory T (TM) cells and TILs rely on fatty acid catabolism to preserve their effector functions due to nutrient competition for glucose with tumor cells. Therefore, enhancing fatty acid catabolism of TILs represents an attractive strategy to increase the efficacy of immunotherapies.Sirt2 is an NAD+ dependent histone deacetylase. We previously showed that upregulation of Sirt2 in human TILs negatively correlates with response to TIL therapy in advanced non-small cell lung cancer (NSCLC) and Sirt2 deficiency leads to hyper-reactive T cells with superior antitumor activity.MethodsSirt2 expression was analyzed by flow cytometry and Western blot. The role of Sirt2 in tumor immunity was studied using in vivo B16F10 tumor challenge models as well as ex vivo analysis including RNA-sequencing, CFSE proliferation assay, DAPI/AnnexinV staining, IFN-γ ELISpot assay, intracellular staining of effector molecules and LDH cytotoxicity assay on WT versus Sirt2KO T cells. Molecular partners of Sirt2 were identified using mass spectrometry (MS) and Co-immunoprecipitation analyses. The role of Sirt2 in T cell metabolism was investigated using seahorse bioanalyzer and LC-MS/MS Metabolomic profiling. AGK2, a Sirt2 selective inhibitor, was used for Sirt2 blockade in human T cells.ResultsSirt2 expression is upregulated during T cell activation, TM stage, and within the TME. Our molecular studies revealed that Sirt2 negatively impacts the acetylation status and the activity of the trifunctional protein, the key enzyme of fatty acid oxidation (FAO). Accordingly, Sirt2 deficiency enhanced FAO and metabolic fitness of activated T cells and mouse TILs isolated from B16F10 tumor nodules. As a consequence of enhanced FAO, Sirt2 deficient mice displayed increased accumulation of TM cells, which was associated with decreased apoptosis and increased survival after tumor challenge leading to superior tumor rejection. Most importantly, pharmacologic inhibition of Sirt2 in human TILs isolated from NSCLC patients enhanced their metabolic fitness and cytotoxic activity against their autologous tumor cells.ConclusionsOur findings indicate Sirt2 as a suppressor of T cell metabolism amenable to therapeutic targeting, and Sirt2 inhibition reprograms T cell metabolic fitness to optimally sustain their effector function within the hypoglycemic TME, thus, leading to an effective anti-tumor immune response.AcknowledgementsThis work was supported in part by K08 CA194273, ACS IRG-17-173-22, NCI Cancer Center Support Grant (P30-CA076292) and the Moffitt Foundation.

Targeting viperin to the mitochondrion inhibits the thiolase activity of the trifunctional enzyme complex

Understanding the mechanisms by which viruses evade host cell immune defenses is important for developing improved antiviral therapies. In an unusual twist, human cytomegalovirus co-opts the antiviral radical SAM enzyme viperin (virus-inhibitory protein, endoplasmic reticulum–associated, interferon-inducible) to enhance viral infectivity. This process involves translocation of viperin to the mitochondrion, where it binds the β-subunit (HADHB) of the mitochondrial trifunctional enzyme complex that catalyzes thiolysis of β-ketoacyl–CoA esters as part of fatty acid β-oxidation. Here we investigated how the interaction between these two enzymes alters their activities and affects cellular ATP levels. Experiments with purified enzymes indicated that viperin inhibits the thiolase activity of HADHB, but, unexpectedly, HADHB activates viperin, leading to synthesis of the antiviral nucleotide 3′-deoxy-3′,4′-didehydro-CTP. Measurements of enzyme activities in lysates prepared from transfected HEK293T cells expressing these enzymes mirrored the findings obtained with purified enzymes. Thus, localizing viperin to mitochondria decreased thiolase activity, and coexpression of HADHB significantly increased viperin activity. Furthermore, targeting viperin to mitochondria also increased the rate at which HADHB is retrotranslocated out of mitochondria and degraded, providing an additional mechanism by which viperin reduces HADHB activity. Targeting viperin to mitochondria decreased cellular ATP levels by more than 50%, consistent with the enzyme disrupting fatty acid catabolism. These results provide biochemical insight into the mechanism by which human cytomegalovirus subverts viperin; they also provide a biochemical rationale for viperin's recently discovered role in regulating thermogenesis in adipose tissues.