MetaboLights MTBLS652 - GNPS Infection-Induced Peroxisome Biogenesis Is a Metabolic Strategy for Herpesvirus Replication
ABSTRACT: Peroxisomes are primarily metabolic organelles with important functions in lipid metabolism, such as fatty acid oxidation and ether phospholipid synthesis (e.g. plasmalogens). Certain viruses, such as human cytomegalovirus (HCMV), hijack organelle functions to facilitate their replication and spread. However, the role of peroxisomes in herpesvirus replication remains elusive. Following a discovery that peroxisome proteins are upregulated upon HCMV infection, we quantified the production of plasmalogens, lipids that require peroxisome functions. In agreement with the increase in peroxisome protein abundance, plasmalogen production was increased by HCMV infection.
Project description:Peroxisomes are primarily metabolic organelles with important functions in lipid metabolism, such as fatty acid oxidation and ether phospholipid synthesis (e.g. plasmalogens). Certain viruses, such as human cytomegalovirus (HCMV), hijack organelle functions to facilitate their replication and spread. However, the role of peroxisomes in herpesvirus replication remains elusive. Following a discovery that peroxisome proteins are upregulated upon HCMV infection, we quantified the production of plasmalogens, lipids that require peroxisome functions. In agreement with the increase in peroxisome protein abundance, plasmalogen production was increased by HCMV infection.
Project description:Peroxisomes are primarily metabolic organelles with important functions in lipid metabolism, such as fatty acid oxidation and ether phospholipid synthesis (e.g., plasmalogens). Certain viruses, such as human cytomegalovirus (HCMV), hijack organelle functions to facilitate their replication and spread. However, the role of peroxisomes in herpesvirus replication remains elusive. Therefore, we used targeted mass spectrometry to quantify 60 peroxisome proteins through the HCMV infection cycle. We provide two proteomic experiments. The first experiment (raw files labeled as 20180123) is of samples in biological triplicate from uninfected human fibroblasts and infected human fibroblasts at 6, 24, 48, 72, and 120 hours post infection. The second experiment (raw files labeled as 201806) is from uninfected/infected fibroblasts during phosphonoformate (PFA) treatment and fibroblasts infected with UV-treated HCMV.
Project description:The ontogeny of the following peroxisomal metabolic pathways was evaluated in mouse liver and brain: alpha-oxidation, beta-oxidation and ether phospholipid synthesis. In mouse embryos lacking functional peroxisomes (PEX5(-/-) knock-out), a deficiency of plasmalogens and an accumulation of the very-long-chain fatty acid C(26:0) was observed in comparison with control littermates, indicating that ether phospholipid synthesis and beta-oxidation are already active at mid-gestation in the mouse. Northern analysis revealed that the enzymes required for the beta-oxidation of straight-chain substrates are present in liver and brain during embryonic development but that those responsible for the degradation of branched-chain substrates are present only in liver from late gestation onwards. The expression pattern of transcripts encoding enzymes of the alpha-oxidation pathway suggested that alpha-oxidation is initiated in the liver around birth and is not active in brain throughout development. Remarkably, a strong induction of the mRNA levels of enzymes involved in alpha-oxidation and beta-oxidation was observed around birth in the liver. In contrast, enzyme transcripts that were expressed in brain were present at rather constant levels throughout prenatal and postnatal development. These results suggest that the defective ether phospholipid synthesis and/or peroxisomal beta-oxidation of straight-chain fatty acids might be involved in the pathogenesis of the prenatal organ defects in peroxisome-deficient mice and men.
Project description:Despite the fact that the discovery of ether-linked phospholipids occurred nearly a century ago, many unanswered questions remain concerning these unique lipids. Here, we characterize the ether-linked lipids of the nematode with HPLC-MS/MS and find that more than half of the phosphoethanolamine-containing lipids are ether-linked, a distribution similar to that found in mammalian membranes. To explore the biological role of ether lipids in vivo, we target fatty acyl-CoA reductase (fard-1), an essential enzyme in ether lipid synthesis, with two distinct RNAi strategies. First, when fard-1 RNAi is initiated at the start of development, the treated animals have severely reduced ether lipid abundance, resulting in a shift in the phosphatidylethanolamine lipid population to include more saturated fatty acid chains. Thus, the absence of ether lipids during development drives a significant remodeling of the membrane landscape. A later initiation of fard-1 RNAi in adulthood results in a dramatic reduction of new ether lipid synthesis as quantified with 15N-tracers; however, there is only a slight decrease in total ether lipid abundance with this adult-only fard-1 RNAi. The two RNAi strategies permit the examination of synthesis and ether lipid abundance to reveal a relationship between the amount of ether lipids and stress survival. We tested whether these species function as sacrificial antioxidants by directly examining the phospholipid population with HPLC-MS/MS after oxidative stress treatment. While there are significant changes in other phospholipids, including polyunsaturated fatty acid-containing species, we did not find any change in ether-linked lipids, suggesting that the role of ether lipids in stress resistance is not through their general consumption as free radical sinks. Our work shows that the nematode will be a useful model for future interrogation of ether lipid biosynthesis and the characterization of phospholipid changes in various stress conditions.
Project description:Peroxisomes play an important role in a variety of metabolic pathways, including the α- and β-oxidation of fatty acids, and the biosynthesis of ether phospholipids. Single peroxisomal enzyme deficiencies (PEDs) are a group of peroxisomal disorders in which either a peroxisomal matrix enzyme or a peroxisomal membrane transporter protein is deficient. To investigate the functional consequences of specific enzyme deficiencies on the lipidome, we performed lipidomics using cultured skin fibroblasts with different defects in the β-oxidation of very long-chain fatty acids, including ABCD1- (ALD), acyl-CoA oxidase 1 (ACOX1)-, D-bifunctional protein (DBP)-, and acyl-CoA binding domain containing protein 5 (ACBD5)-deficient cell lines. Ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry revealed characteristic changes in the phospholipid composition in fibroblasts with different fatty acid β-oxidation defects. Remarkably, we found that ether phospholipids, including plasmalogens, were decreased. We defined specific phospholipid ratios reflecting the different enzyme defects, which can be used to discriminate the PED fibroblasts from healthy control cells.
Project description:In recent years, much progress has been made with respect to the unravelling of the functions of peroxisomes in metabolism, and it is now well established that peroxisomes are indispensable organelles, especially in higher eukaryotes. Peroxisomes catalyse a number of essential metabolic functions including fatty acid beta-oxidation, ether phospholipid biosynthesis, fatty acid alpha-oxidation and glyoxylate detoxification. The involvement of peroxisomes in these metabolic pathways necessitates the transport of metabolites in and out of peroxisomes. Recently, considerable progress has been made in the characterization of metabolite transport across the peroxisomal membrane. Peroxisomes posses several specialized transport systems to transport metabolites. This is exemplified by the identification of a specific transporter for adenine nucleotides and several half-ABC (ATP-binding cassette) transporters which may be present as hetero- and homo-dimers. The nature of the substrates handled by the different ABC transporters is less clear. In this review we will describe the current state of knowledge of the permeability properties of the peroxisomal membrane.
Project description:Viral proteins have evolved to target cellular organelles and usurp their functions for virus replication. Despite the knowledge of these critical functions for several organelles, little is known about peroxisomes during infection. Peroxisomes are primarily metabolic organelles with important functions in lipid metabolism. Here, we discovered that the enveloped viruses human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) induce the biogenesis of and unique morphological changes to peroxisomes to support their replication. Targeted proteomic quantification revealed a global virus-induced upregulation of peroxisomal proteins. Mathematical modeling and microscopy structural analysis show that infection triggers peroxisome growth and fission, leading to increased peroxisome numbers and irregular disc-like structures. HCMV-induced peroxisome biogenesis increased the phospholipid plasmalogen, thereby enhancing virus production. Peroxisome regulation and dependence were not observed for the non-enveloped adenovirus. Our findings uncover a role of peroxisomes in viral pathogenesis, with likely implications for multiple enveloped viruses.
Project description:Ozone is a common environmental toxicant to which individuals are exposed to on a daily basis. While biochemical end points such as increased mortality, decrements in pulmonary function, and initiation of inflammatory processes are known, little is actually understood regarding the chemical mechanisms underlying changes in pulmonary health, especially for low concentrations of ozone. This study was undertaken to investigate ozone-induced oxidation of endogenous lipids that are potentially exposed to environmental ozone within lung, specifically focusing on plasmalogen glycerophospholipids present in pulmonary surfactant. Sensitive liquid chromatography-mass spectrometry methods were developed to follow oxidation of diacyl and plasmalogen phosphatidylethanolamine (PE) phospholipids and to identify and quantitate products generated by ozonolysis. Using a unilamellar vesicle system containing a 1:1 molar mixture of 1-O-octadec-1'-enyl-2-octadecenoyl-PE and 1,2-dihexadecanoyl-PC, these studies revealed that the vinyl ether bond of plasmalogens was oxidized preferentially at low concentrations of ozone (100 ppb), when compared to olefinic bond oxidation on omega-9 of the fatty acyl chain in the same phospholipids. Major phospholipid products generated were identified as 1-formyl-2-octadecenoyl-PE and 1-hydroxy-2-octadecenoyl-PE. Heptadecanal and heptadecanoic acid production was also quantitated using gas chromatography-mass spectrometry, and production was consistent with oxidation of the vinyl ether, at low concentrations of ozone. Analysis of murine lung surfactant from C57Bl/6 mice revealed several plasmalogen PE lipid species, encompassing approximately 38% of total PE species. Upon exposure of ozone (0 and 100 ppb) to murine surfactant, plasmalogen PE molecular species preferentially reacted, as compared to diacyl PE molecular species. Lysophospholipids, pentadecanal, and nonanal were found to be the primary products of surfactant ozone oxidation.
Project description:Choline is an essential nutrient required for normal neuronal and muscular development, as well as homeostatic regulation of hepatic metabolism. In the liver, choline is incorporated into the main eukaryotic phospholipid, phosphatidylcholine (PC), and can enter one-carbon metabolism via mitochondrial oxidation. Hepatitis C virus (HCV) is a hepatotropic positive-strand RNA virus that similar to other positive-strand RNA viruses and can impact phospholipid metabolism. In the current study we sought to interrogate if HCV modulates markers of choline metabolism following in vitro infection, while subsequently assessing if the inhibition of choline uptake and metabolism upon concurrent HCV infection alters viral replication and infectivity. Additionally, we assessed whether these parameters were consistent between cells cultured in fetal bovine serum (FBS) or human serum (HS), conditions known to differentially affect in vitro HCV infection. We observed that choline transport in FBS- and HS-cultured Huh7.5 cells is facilitated by the intermediate affinity transporter, choline transporter-like family (CTL). HCV infection in FBS, but not HS-cultured cells diminished CTL1 transcript and protein expression at 24 h post-infection, which was associated with lower choline uptake and lower incorporation of choline into PC. No changes in other transporters were observed and at 96 h post-infection, all differences were normalized. Reciprocally, limiting the availability of choline for PC synthesis by use of a choline uptake inhibitor resulted in increased HCV replication at this early stage (24 h post-infection) in both FBS- and HS-cultured cells. Finally, in chronic infection (96 h post-infection), inhibiting choline uptake and metabolism significantly impaired the production of infectious virions. These results suggest that in addition to a known role of choline kinase, the transport of choline, potentially via CTL1, might also represent an important and regulated process during HCV infection.
Project description:The mechanism by which type II interferon (IFN) inhibits virus replications remains to be identified. Murine leukemia virus (MLV) replication was significantly restricted by γ-IFN, but not human immunodeficiency virus type 1 (HIV-1) replication. Because MLV enters host cells via endosomes, we speculated that certain cellular factors among γ-IFN-induced, endosome-localized proteins inhibit MLV replication. We found that γ-IFN-inducible lysosomal thiolreductase (GILT) significantly restricts HIV-1 replication as well as MLV replication by its thiolreductase activity. GILT silencing enhanced replication-defective HIV-1 vector infection and virion production in γ-IFN-treated cells, although γ-IFN did not inhibit HIV-1 replication. This result showed that GILT is required for the anti-viral activity of γ-IFN. Interestingly, GILT protein level was increased by γ-IFN in uninfected cells and env-deleted HIV-1-infected cells, but not in full-length HIV-1-infected cells. γ-IFN-induced transcription from the γ-IFN-activation sequence was attenuated by the HIV-1 Env protein. These results suggested that the γ-IFN cannot restrict HIV-1 replication due to the inhibition of γ-IFN signaling by HIV-1 Env. Finally, we found that 4,4'-dithiodipyridine (4-PDS), which inhibits S-S bond formation at acidic pH, significantly suppresses HIV-1 vector infection and virion production, like GILT. In conclusion, this study showed that GILT functions as a host restriction factor against the retroviruses, and a GILT mimic, 4-PDS, is the leading compound for the development of novel concept of anti-viral agents.