The p97-UBXD8 complex regulates ER-Mitochondria contact sites by altering membrane lipid saturation and composition.

The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-Mitochondria contact sites (ERMCS) is a platform for critical cellular processes, particularly lipid synthesis. How contacts are remodeled and the impact of altered contacts on lipid metabolism remains poorly understood. We show that the p97 AAA-ATPase and its adaptor ubiquitin-X domain adaptor 8 (UBXD8) regulate ERMCS. The p97-UBXD8 complex localizes to contacts and its loss increases contacts in a manner that is dependent on p97 catalytic activity. Quantitative proteomics and lipidomics of ERMCS demonstrates alterations in proteins regulating lipid metabolism and a significant change in membrane lipid saturation upon UBXD8 deletion. Loss of p97-UBXD8 increased membrane lipid saturation via SREBP1 and the lipid desaturase SCD1. Aberrant contacts can be rescued by unsaturated fatty acids or overexpression of SCD1. We find that the SREBP1-SCD1 pathway is negatively impacted in the brains of mice with p97 mutations that cause neurodegeneration. We propose that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation.

Human Cytomegalovirus Uses a Host Stress Response To Balance the Elongation of Saturated/Monounsaturated and Polyunsaturated Very-Long-Chain Fatty Acids.

Stress and virus infection regulate lipid metabolism. Human cytomegalovirus (HCMV) infection induces fatty acid (FA) elongation and increases the abundance of lipids with very-long-chain FA (VLCFA) tails. While reprogramming of metabolism can be stress related, the role of stress in HCMV reprogramming of lipid metabolism is poorly understood. In this study, we engineered cells to knock out protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) in the ER stress pathway and measured lipid changes using lipidomics to determine if PERK is needed for lipid changes associated with HCMV infection. In HCMV-infected cells, PERK promotes increases in the levels of phospholipids with saturated FA (SFA) and monounsaturated FA (MUFA) VLCFA tails. Further, PERK enhances FA elongase 7 (ELOVL7) protein levels, which elongates SFA and MUFA VLCFAs. Additionally, we found that increases in the elongation of polyunsaturated fatty acids (PUFAs) associated with HCMV infection were independent of PERK and that lipids with PUFA tails accumulated in HCMV-infected PERK knockout cells. Additionally, the protein levels of ELOVL5, which elongates PUFAs, are increased by HCMV infection through a PERK-independent mechanism. These observations show that PERK differentially regulates ELOVL7 and ELOVL5, creating a balance between the synthesis of lipids with SFA/MUFA tails and PUFA tails. Additionally, we found that PERK was necessary for virus replication and the infectivity of released viral progeny. Overall, our findings indicate that PERK-and, more broadly, ER stress-may be necessary for the membrane biogenesis needed to generate infectious HCMV virions.IMPORTANCE HCMV is a common herpesvirus that establishes lifelong persistent infections. While infection is asymptomatic in most people, HCMV causes life-threatening illnesses in immunocompromised people, including transplant recipients and cancer patients. Additionally, HCMV infection is a leading cause of congenital disabilities. HCMV replication relies on lipid synthesis. Here, we demonstrated that the ER stress mediator PERK controls FA elongation and the cellular abundance of several types of lipids following HCMV infection. Specifically, PERK promotes FA elongase 7 synthesis and phospholipids with saturated/monounsaturated very-long-chain FA tails. Overall, our study shows that PERK is an essential host factor that supports HCMV replication and promotes lipidome changes caused by HCMV infection.

Hypoxia-Inducible Factor 1α (HIF1α) Suppresses Virus Replication in Human Cytomegalovirus Infection by Limiting Kynurenine Synthesis.

Human cytomegalovirus (HCMV) replication depends on the activities of several host regulators of metabolism. Hypoxia-inducible factor 1α (HIF1α) was previously proposed to support virus replication through its metabolic regulatory function. HIF1α protein levels rise in response to HCMV infection in nonhypoxic conditions, but its effect on HCMV replication was not investigated. We addressed the role of HIF1α in HCMV replication by generating primary human cells with HIF1α knocked out using CRISPR/Cas9. When HIF1α was absent, we found that HCMV replication was enhanced, showing that HIF1α suppresses viral replication. We used untargeted metabolomics to determine if HIF1α regulates metabolite concentrations in HCMV-infected cells. We discovered that in HCMV-infected cells, HIF1α suppresses intracellular and extracellular concentrations of kynurenine. HIF1α also suppressed the expression of indoleamine 2,3-dioxygenase 1 (IDO1), the rate-limiting enzyme in kynurenine synthesis. In addition to its role in tryptophan metabolism, kynurenine acts as a signaling messenger by activating aryl hydrocarbon receptor (AhR). Inhibiting AhR reduces HCMV replication, while activating AhR with an exogenous ligand increases virus replication. Moreover, we found that feeding kynurenine to cells promotes HCMV replication. Overall, our findings indicate that HIF1α reduces HCMV replication by regulating metabolism and metabolite signaling.IMPORTANCE Viruses, including human cytomegalovirus (HCMV), reprogram cellular metabolism using host metabolic regulators to support virus replication. Alternatively, in response to infection, the host can use metabolism to limit virus replication. Here, our findings show that the host uses hypoxia-inducible factor 1α (HIF1α) as a metabolic regulator to reduce HCMV replication. Further, we found that HIF1α suppresses kynurenine synthesis, a metabolite that can promote HCMV replication by signaling through the aryl hydrocarbon receptor (AhR). In infected cells, the rate-limiting enzyme in kynurenine synthesis, indoleamine 2,3-dioxygenase 1 (IDO1), is suppressed by a HIF1α-dependent mechanism. Our findings describe a functional connection between HIF1α, IDO1, and AhR that allows HIF1α to limit HCMV replication through metabolic regulation, advancing our understanding of virus-host interactions.

Sulfation of hesperetin, naringenin and apigenin by the human cytosolic sulfotransferases: a comprehensive analysis.

Previous studies have revealed sulfation as a major pathway for the metabolism of hesperetin, naringenin and apigenin. The current study was designed to identify the human cytosolic sulfotransferase (SULT) enzyme(s) capable of sulfating these flavonoid compounds. Of the thirteen human SULTs, six (1A1, 1A2, 1A3, 1B2, 1C4, 1E1) displayed significant sulfating activity toward hesperetin, five (1A1, 1A2, 1A3, 1B2, 1C4) displayed sulfating activity towards naringenin, and four (1A1, 1A2, 1A3, 1C4) showed sulfating activity towards apigenin. Of the four human organ specimens tested, liver and intestine cytosols displayed much higher hesperetin-, naringenin- and apigenin-sulfating activity than lung and kidney cytosols. Moreover, sulfation of hesperetin, naringenin and apigenin was shown to take place in HepG2 human hepatoma cells and Caco-2 human colon adenocarcinoma cells under cultured conditions. Taken together, these results provided a biochemical basis underlying the metabolism of hesperetin, naringenin and apigenin through sulfation in humans.[Formula: see text].

Human Cytomegalovirus pUL37x1 Is Important for Remodeling of Host Lipid Metabolism.

Human cytomegalovirus (HCMV) replication requires host metabolism. Infection alters the activity in multiple metabolic pathways, including increasing fatty acid elongation and lipid synthesis. The virus-host interactions regulating the metabolic changes associated with replication are essential for infection. While multiple host factors, including kinases and transcription factors, important for metabolic changes that occur following HCMV infection have been identified, little is known about the viral factors required to alter metabolism. In this study, we tested the hypothesis that pUL37x1 is important for the metabolic remodeling that is necessary for HCMV replication using a combination of metabolomics, lipidomics, and metabolic tracers to measure fatty acid elongation. We observed that fibroblast cells infected with wild-type (WT) HCMV had levels of metabolites similar to those in cells infected with a mutant virus lacking the UL37x1 gene, subUL37x1. However, we found that relative to WT-infected cells, subUL37x1-infected cells had reduced levels of two host proteins that were previously demonstrated to be important for lipid metabolism during HCMV infection: fatty acid elongase 7 (ELOVL7) and the endoplasmic reticulum (ER) stress-related kinase PERK. Moreover, we observed that HCMV infection results in an increase in phospholipids with very-long-chain fatty acid tails (PL-VLCFAs) that contain 26 or more carbons in one of their two tails. The levels of many PL-VLCFAs were lower in subUL37x1-infected cells than in WT-infected cells. Overall, we conclude that although pUL37x1 is not necessary for network-wide metabolic changes associated with HCMV infection, it is important for the remodeling of a subset of metabolic changes that occur during infection.IMPORTANCE Human cytomegalovirus (HCMV) is a common pathogen that asymptomatically infects most people and establishes a lifelong infection. However, HCMV can cause end-organ disease that results in death in the immunosuppressed and is a leading cause of birth defects. HCMV infection depends on host metabolism, including lipid metabolism. However, the viral mechanisms for remodeling of metabolism are poorly understood. In this study, we demonstrate that the viral UL37x1 protein (pUL37x1) is important for infection-associated increases in lipid metabolism, including fatty acid elongation to produce very-long-chain fatty acids (VLCFAs). Furthermore, we found that HCMV infection results in a significant increase in phospholipids, particularly those with VLCFA tails (PL-VLCFAs). We found that pUL37x1 was important for the high levels of fatty acid elongation and PL-VLCFA accumulation that occur in HCMV-infected cells. Our findings identify a viral protein that is important for changes in lipid metabolism that occur following HCMV infection.

Sulfate conjugation of daphnetin by the human cytosolic sulfotransferases.

ETHNOPHARMACOLOGICAL RELEVANCE: In Turkey, daphnetin-containing Daphne oleoides is used as a folk medicine for treating rheumatic pain and lumbago. A daphnetin-containing traditional Chinese medicine tablet, named Zushima-Pian, is available in China for treating rheumatoid arthritis. The present study aimed to investigate the metabolism of daphnetin through sulfation in cultured human cells and to identify the human cytosolic sulfotransferase(s) (SULT(s)) that is(are) capable of mediating the sulfation of daphnetin. MATERIALS AND METHODS: Cultured HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells were labeled with [(35)S]sulfate in the presence of different concentrations of daphnetin. Thirteen known human SULTs, previously expressed and purified, as well as cytosols of human kidney, liver, lung, and small intestine, were examined for daphnetin-sulfating activity using an established sulfotransferase assay. RESULTS: [(35)S]sulfated daphnetin was found to be generated and released by HepG2 cells and Caco-2 cells labeled with [(35)S] sulfate in the presence of daphnetin. Among the 13 known human SULTs, SULT1A1, SULT1A2, SULT1A3, SULT1B1, and SULT1C4 displayed significant sulfating activity toward daphnetin. Of the four human organ samples later tested, small intestine and liver cytosols displayed considerably higher daphnetin-sulfating activity than those of lung and kidney. CONCLUSION: The results derived from the present study showed unequivocally that daphnetin could be sulfated in cultured human cells and by purified human SULT enzymes as well as human organ cytosols. The information obtained provided a basis for further studies on the metabolism of daphnetin through sulfation in vivo.

Role of SULT-mediated sulfation in the biotransformation of 2-butoxyethanol and sorbitan monolaurate: A study using zebrafish SULTs.

2-Butoxyethanol and sorbitan monolaurate are major components of oil dispersants that are applied in large quantities to control oil spill in the aquatic environment. An important question is whether aquatic animals are equipped with mechanisms for the detoxification of these oil dispersant compounds. The current study aimed to examine whether zebrafish cytosolic sulfotransferases (SULTs) are capable of sulfating 2-butoxyethanol and sorbitan monolaurate. A systematic analysis of 18 zebrafish SULTs revealed that SULT3 ST1 showed the strongest sulfating activity toward 2-butoxyethanol, while SULT1 ST3 displayed the strongest sulfating activity toward sorbitan monolaurate. The pH-dependence of these two SULTs in mediating the sulfation of 2-butoxyethanol or sorbitan monolaurate was examined. Taken together, these results implied that SULT-mediated sulfation may function in the detoxification of these two oil dispersant compounds.

Sulfation of 6-Gingerol by the Human Cytosolic Sulfotransferases: A Systematic Analysis.

Previous studies have demonstrated the presence of the sulfated form of 6-gingerol, a major pharmacologically active component of ginger, in plasma samples of normal human subjects who were administered 6-gingerol. The current study was designed to systematically identify the major human cytosolic sulfotransferase enzyme(s) capable of mediating the sulfation of 6-gingerol. Of the 13 known human cytosolic sulfotransferases examined, six (SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C4, SULT1E1) displayed significant sulfating activity toward 6-gingerol. Kinetic parameters of SULT1A1, SULT1A3, SULT1C4, and SULT1E1 that showed stronger 6-gingerol-sulfating activity were determined. Of the four human organ samples tested, small intestine and liver cytosols displayed considerably higher 6-gingerol-sulfating activity than those of the lung and kidney. Moreover, sulfation of 6-gingerol was shown to occur in HepG2 human hepatoma cells and Caco-2 human colon adenocarcinoma cells under the metabolic setting. Collectively, these results provided useful information relevant to the metabolism of 6-gingerol through sulfation both in vitro and in vivo.