Hepatocellular carcinoma is highly refractory cancer which is resistant to conventional chemotherapy and radiotherapy, carrying a dismal prognosis. Although many anticancer drugs have been developed for treating HCC, sorafenib is the only effective treatment, but it only prolongs survival duration for about 3 months. Recently, oncolytic virotherapy has shown promising results in treating HCCs and the effects can be more enhanced by adopting immune modulatory molecules. This review discusses the current status of treating HCC and the effective strategy of oncolytic virus-based immunotherapy for the treatment of HCCs. Hepatocellular carcinoma (HCC) is the third most common cancer with a leading cause of cancer-related death worldwide and is the only carcinoma with increasing mortality , 2. The Therefore, HCC is considered a highly refractory cancer and is resistant to conventional chemotherapy. To overcome these issues, the focus is shifting from antiangiogenic therapy  to novelThis review discusses the current status and the barriers of immunotherapy against HCC and effective immunotherapy strategies using oncolytic virus for HCC.Treatment options for HCC are divided into three categories as follows: (1) surgical therapies , (2) liver-directed nonsurgical therapies , radio frequency ablation (RFA), TACE, radiation, and radioembolization), or (3) systemic nonsurgical therapies . Since surgical therapies and liver-directed surgical therapies are generally used in the early stages of HCCs and HCC is typically diagnosed late in the course of patient with chronic liver diseases , here weDoxorubicin is the most studied chemotherapy agent for advanced HCC. Among the agents tried, doxorubicin-based regimens appear to have the greatest efficacy with response rates of 20–30% and a minimal impact on survival . Mitoxan5-Fluorouracil (5-FU) has broad antitumor efficacy with acceptably low toxicity. Although the response rate with only 5-FU has been low, combination with leucovorin can bring response rates to as high as 28% .Gemcitabine has modest activity at best. It is reported that 5 of 28 patients that received gemcitabine had a partial response of short duration (~13 weeks) . IrinoteCisplatin-based combination therapy appears to show higher objective response rates than that in noncisplatin-based therapy, although it is not clear whether it confers a survival benefit. Cisplatin plus doxorubicin had 18 and 49% objective responses, respectively , 36. TheTreatment approaches are now directed against a specific molecular defect, which is termed “molecular-targeted therapies.” Although the molecular pathogenesis of HCC remains poorly understood, the following signaling pathways are notably involved: (1) the epidermal growth factor receptor (EGFR)/EGF (HER1) in the carcinogenesis and proliferative behavior of HCC –49. (2) Oncolytic viruses (OVs) have been recently recognized as an effective treatment for cancer in preclinical models and promising clinical responses in human cancer patients . OVs havMost of the preclinical studies use adenoviruses and vesicular stomatitis virus (VSV) as the vector, due to their natural ability to kill or target HCC cells. For instance, adenoviruses have excellent tropism towards hepatocytes , in caseE1A and E1B genes of adenovirus have made it as oncolytic vector against the cancer cells [E1A gene is responsible for inactivation of several proteins, including retinoblastoma, allowing entry into S-phase [Adenoviruses are nonenveloped viruses, consist of double-stranded DNA (dsDNA) about 36 kb in size. There are seven subgroups reported, based on cross-reactivity patterns of neutralizing antibodies, from A to G. These are further classified as serotypes. As of now, 57 serotypes have been reported. The pathogenicity and tissue tropism are similar within the subgroups . Adenovier cells . The E1A S-phase . The E1B S-phase  whereas E1b gene, was engineered. The efficacy of this virus was checked in HCC cell lines and animal models in terms of tumor targeting, which showed selective gene expression, tumor regression, and prolonged survival period. The results compared with those of CNHK300 confirmed antitumor efficacy and selective replication of CNHK500 in HCC models [In order to overcome to this hurdle, adenovirus vector, ZD55, was constructed with -Smac (second mitochondria-derived activator of caspases)/TRAIL as ZD55-Smac/ZD55-TRAIL. The combined antitumor effect of these vectors was evaluated in mice xenograft models and cell lines. A significant regression of tumor size was reported in contrast to partial effect of single treatment of ZD55-Smac or ZD55-TRAIL. Moreover, in vitro-based analysis showed activation of caspases and significant reduction of X-linked inhibitor of apoptosis protein (XIAP) expression . VariousC models .β was generated by homologous recombination of ZD55 vector with interferon-β (IFN-β) containing vector. The antitumor efficacy and IFN-β expression of ZD55-IFN-β were evaluated in HCC cell lines and mice xenograft model. In contrast to Ad5-IFN-β-treated model, 100% cytopathic effect was examined in cell lines and more IFN-β expression were observed in both ZD55-IFN-β received mice and cell lines. This study confirmed the ability of adenovirus as a carrier of potential anticancer genes [Another viral strain, a monoregulated adenovirus CNHK300, was constructed with hTERT, and its antitumor efficacy was evaluated in different cell lines. The detection of hTERT expression and cytotoxicity, especially at low multiplicity of infection (MOI), confirmed the oncolytic activity of CNHK300 . ZD55-IFer genes .let7T was constructed as adenovirus with microRNA (miRNA), let-7. It was generated as introducing eight copies of let-7 target sites (let7T) into the 39 untranslated regions of E1A. A higher selective replication and cytotoxicity were reported in HCC cell lines when compared to normal liver cell lines. These were also verified in animal models [SG7011l models . Telomell models .γ (IFN-γ) production were also reported in this study [α-fetoprotein- (AFP-) E1A-IRES-E1B bicistronic cassette derived from Ad5, CV890 showed a synergic antitumor effect in vitro as well as in vivo [Adenoviruses armed with apoptosis-induced and immune-stimulatory molecules, such as human TRAIL and IL-12, were constructed as Ad-ΔB/TRAIL and Ad-ΔB/IL-12, respectively. The combined antitumor effects of these vectors were evaluated in Hep3B and HuH7 HCC cell lines. In addition, in vivo efficacy was examined in transplanted Hep3B-orthotopic model. A significant reduction in tumor size of orthotopic model and increased HCC cell death in cell culture were reported. Interestingly, this study documented a remarkable suppression of VEGF with infiltration of natural killer cells (NK cells) and antigen-presenting cells (APCs) at tumor microenvironment (TME). Moreover, enhanced apoptosis and upregulation of interferon-is study . In addi in vivo .Herpes simplex viruses (HSV) consist of dsDNA (154 kb) as genetic material. Cell receptors, such as herpesvirus entry mediator (HVEM), nectin 1, and nectin 2, are used for cell entry. Using HSV as an oncolytic agent, only very few studies were conducted in HCC model. In order to enhance HCC cell-specific tropism, liver-cancer-specific oncolytic virus (LCSOV) was created. It was constructed by linking the essential viral glycoprotein H gene with the liver specific apolipoprotein E (apoE)-AAT promoter. Then, miR-122a complimentary sequence was further inserted to the 3′ untranslated region (3′UTR). In addition, let-7 was also engineered into the same 3′UTR to increase the safety of this virus. The highly selective replication and tumor cell lysis were reported in xenograft as well as in cell culture models . HSV, G4TK)  . Intratuft model .β-gal into Buffalo rats bearing orthotopically implanted multifocal HCC showed efficient viral transduction, tumor-selective viral replication, and extensive oncolysis. There were no significant vector-associated toxicities and damage to the hepatic parenchyma reported, whereas prolonged survival was reported in vector-treated group [α/β) was checked in rVSV-NDV/F- (L289A-) treated HCC cell lines and rat models. Potential replication efficacy and no toxicity were documented as major outcomes. This study reported an increased index of oncolytic VSV virotherapy in interferon-treated advanced HCC models [VSV consists of single strand RNA (ssRNA) as a genetic material. Due to its intrinsic infectivity against cancer cells, most of the earlier preclinical studies used VSV for oncolytic virotherapy. VSV was recombined with green fluorescence protein (GFP) to examine antitumor efficacy and toxicity in HCC cell panels. The efficient selective replication and cytotoxicity were documented. IT injection of the virus in rat liver showed tumor destruction and tumor growth inhibition, which leads to prolonged survival of animals . Hepaticed group . Recombied group . AntivirC models . In ordeC models .The effect of the combination of an oncolytic MeV with the novel oral HDACi resminostat (Res) was checked in HCC cell panels. The combination effect showed a boosted cytotoxic effect as an enhanced induction of apoptosis with improved rate of primary infections . In ordedI1520 vector [9 pfu [Despite a number of OVs were tried in the preclinical studies, only a few among them have entered into the clinical studies . The rea0 vector . Most of0 vector , 81, 92.r [9 pfu . In order [9 pfu .A sequential therapy of JX-594, followed by sorafenib, a multikinase inhibitor and antagonist to vascular endothelial growth factor receptor (VEGFR) in three HCC patients, showed well-tolerance, associated with significantly decreased tumor perfusion and tumor responses  . In addiDue to limitations in conventional therapies for HCC, new treatment approaches have been established using modern molecular techniques. Oncolytic virotherapy is using oncolytic viruses which selectively infect and kill cancer cells. In order to enhance immunity against cancer cells, genes elevating onco-immunity have been engineered within oncolytic virus. Past and ongoing clinical trials have investigated anticancer efficacy and toxicities of oncolytic viruses, particularly JX-594 (GM-CSF encoding VV) for HCC treatment. GM-CSF as an immune-stimulatory cytokine boosts host immune activity through the infiltration of dendritic cells (DCs) and CD4+ and CD8+ T cells at tumor sites. Despite these outcomes have shown favorable results for prognosis of HCC treatment, as an immunotherapeutic approach, more advanced methods, that is, combination therapy with immune checkpoint inhibitors (ICIs) such as monoclonal antibodies (mAbs) against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed death-ligand 1 (PD-L1), and/or programmed cell death protein 1(PD-1), would be taken into the account . This is


Proper expression of key reproductive hormones from gonadotrope cells of the pituitary is required for pubertal onset and reproduction. To further our understanding of the molecular events taking place during embryonic development, leading to expression of the glycoproteins luteinizing hormone (LH) and follicle-stimulating hormone (FSH), we characterized chromatin structure changes, imparted mainly by histone modifications, in model gonadotrope cell lines.Cga), αT3–1 , and LβT2 , to assess changes in chromatin status and transcription factor access of gonadotrope-specific genes.We evaluated chromatin status and gene expression profiles by chromatin immunoprecipitation assays, DNase sensitivity assay, and RNA sequencing in three developmentally staged gonadotrope cell lines, αT1–1 (progenitor, expressing Cga, to have an open chromatin conformation in all three cell lines. In contrast, chromatin status of Gnrhr is open only in αT3–1 and LβT2 cells. Lhb begins to open in LβT2 cells and was further opened by activin treatment. Histone H3 modifications associated with active chromatin were high on Gnrhr in αT3–1 and LβT2, and Lhb in LβT2 cells, while H3 modifications associated with repressed chromatin were low on Gnrhr, Lhb, and Fshb in LβT2 cells. Finally, chromatin status correlates with the progressive access of LHX3 to Cga and Gnrhr, followed by PITX1 binding to the Lhb promoter.We found the common mRNA α-subunit of LH and FSH, called Cga, Gnrhr, Lhb, and Fshb are not only controlled by developmental transcription factors, but also by epigenetic mechanisms that include the modulation of chromatin structure, and histone modifications.Our data show the gonadotrope-specific genes  Cga, glycoprotein hormones also known as αGSU) is detectable at E11.5 in the mouse, followed by the nuclear receptor Steroidogenic Factor 1 (Sf1 or Nr5a1), and the gonadotropin-releasing hormone receptor (Gnrhr). Finally, the gonadotropin hormone beta subunit mRNAs emerge at E16.5 for Lhb and E17.5 for Fshb . In. InCga. Cga, Gnrhr, Lhb, and Fshb in these model cell lines and demonstrate that these promoters are not only controlled by developmental transcription factors, but also by epigenetic mechanisms that include the modulation of chromatin structure, and histone modifications.Our data show exciting new evidence of gonadotrope-specific chromatin changes taking place during development using immortalized model gonadotrope cell lines. Although the cell lines utilized remain an in vitro system, representative of developing gonadotrope cell lines, we show a progressive opening of the gonadotrope-specific gene promoters,


The H6PD gene is amplified in several types of malignancies, and earlier work pointed toward a potential involvement of the enzyme in cancer cell growth. In the present study, we demonstrated a pivotal role of H6PD in proliferation and migratory potential of 3 human breast cancer cell lines. Knockdown of H6PD decreased proliferation and migration in SUM159, MCF7, and MDA-MB-453 cells. To understand the mechanism through which H6PD exerts its effects, we investigated the cellular changes after H6PD silencing in SUM159 cells. Knockdown of H6PD resulted in an increase in ER lumen oxidation, and down-regulation of many components of the unfolded protein response, including the transcription factors activating transcription factor-4, activating transcription factor-6, split X-box binding protein-1, and CCAAT/enhancer binding protein homologous protein. This effect was accompanied by an increase in sarco/endoplasmic reticulum Ca2+-ATPase-2 pump expression and an decrease in inositol trisphosphate receptor-III, which led to augmented levels of calcium in the ER. Further characterization of the molecular pathways involving H6PD could greatly broaden our understanding of how the ER microenvironment sustains malignant cell growth.—Tsachaki, M., Mladenovic, N., Štambergová, H., Birk, J., Odermatt, A. Hexose-6-phosphate dehydrogenase controls cancer cell proliferation and migration through pleiotropic effects on the unfolded protein response, calcium homeostasis, and redox balance.Hexose-6-phosphate dehydrogenase (H6PD) produces reduced NADPH in the endoplasmic reticulum (ER) lumen. NADPH constitutes a cofactor for many reducing enzymes, and its inability to traverse biologic membranes makes  In addition, the levels of the UPR mediator split X-box-binding protein (sXBP)-1 were increased, and microarray analysis showed altered expression of several UPR proteins. Furthermore, muscles from H6PD−/− mice showed decreased expression of the sarco/ER Ca2+-ATPase (SERCA), which is responsible for pumping calcium from the cytosol into the ER at the expense of ATP.Hexose-6-phosphate dehydrogenase (H6PD)  is the oysiology , 8. MuscH6PD gene is amplified in 3–4% of pancreatic, sarcomatous, and ovarian tumors and in 1–2% of breast, lung adenocarcinoma, and melanoma tumors, supporting a role of the enzyme in cancer cell growth.Recently, down-regulation of H6PD with short interfering (si)RNAs in the murine cancer cell lines CT26 (colon) and 4T1 (breast) correlated with a reduction in the overall glucose uptake, as well as a decreased cell proliferation . In addi−), estrogen receptor (ER−), nor the Her2 receptor (Her2−)], the PR+, ER+, Her2− cell line MCF7, and the PR−, ER−, Her2+ cell line MDA-MB-453. We further demonstrated that H6PD knockdown dramatically reduces migration in all cell lines tested. Subsequently, we attempted to elucidate the mechanism through which H6PD influences cancer cell growth. Our results suggest a major role of H6PD in regulating UPR signaling proteins, as well as ER calcium balance. H6PD depletion also caused an increase in ER oxidation and a significant decrease in cellular oxygen consumption rate. The above findings highlight, for the first time, the consequences of NADPH depletion within the ER for cancer cell physiology, paving the way for further investigations into the ER-related molecular pathways that promote malignant cell proliferation and migration.In the present study, we showed that H6PD promotes cancer cell proliferation by using 3 different breast cancer cell models, each representing one of the main molecular subtypes of breast cancer: the triple-negative cell line SUM159 , followed by incubation for 1 h at 37°C. After 3 washes with HEPES buffer, fluorescence was monitored in real time using an inverted microscope . Once the signal was stabilized, 10 mM carbachol (Cch) was added, and fluorescence was monitored until return to baseline. For fluorescence intensity measurements, an entire field visualized under the ×20 objective was chosen, and signal was quantified with Suite X software .2 before transfer to the XF96 instrument. Three measurements of OCR and ECAR were performed before injection of 1 μM oligomycin and 0.5 μM FCCP, followed by 5 more measurements. During the experiment, the cells were kept in a humidified chamber at 37°C, under normal oxygen conditions. Data were processed using the Wave 2.0 software and the XF Cell Energy phenotype report generator (Agilent Technologies).The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in real-time using the Seahorse XF96 analyzer (Agilent Technologies). Cell line characterization was performed according to the manufacturer’s instructions, to achieve optimal cell densities and determine the concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) to be applied. Cells were transfected with siRNAs and seeded in 6 replicates for each sample on a 96-well microplate. On the day of the measurement, cells were washed twice with XF base medium (Agilent Technologies) and supplemented with the appropriate amounts of pyruvate, glutamine, and glucose, according to the culture medium concentrations of each cell line, and the pH was adjusted to 7.4. The cells were incubated in this medium for 1 h in an incubator without COet al. . Significance was accepted at P < 0.05.Data are presented as means ± To examine the impact of H6PD on cancer cell proliferation, we first silenced its expression by using specific siRNAs in the SUM159, MCF7, and MDA-MB-453 cell lines. Subsequently, we performed Western blot analysis at 24, 48, and 72 h after siRNA delivery, to evaluate the efficiency of protein level reduction over time . In SUM1After confirming the knockdown efficiency in our cell models, we down-regulated H6PD and monitored cell proliferation with the xCelligence RTCA. The transfection procedure did not affect proliferation, as observed when comparing nontransfected and mock siRNA-transfected cells A. To inv2/mitotic CCNB1 were greatly reduced after H6PD down-regulation  and p27 (gene CDKN1B), which control progression of the cell cycle at the G1 checkpoint, were increased. In accordance, cell cycle analysis at 48 h after H6PD down-regulation, showed a higher percentage of cells at the G1 phase , CCNA2 (regulating S phase and mitotic entry), and Ggulation A. The leG1 phase B. IncreaBesides increased proliferation potential, a central aspect of tumor cell physiology is their ability to migrate and invade distant tissues. To evaluate whether H6PD is involved in cancer cell migration, we performed a cell migration assay (Transwell) with a serum gradient as a chemoattractant. H6PD knockdown led to an 80% decrease in migration of SUM159 cells, which had the highest migratory potential of the 3 cell lines analyzed (CTNNB1), which was also found to be down-regulated -1α, which, upon activation and phosphorylation, leads to the splicing of the sXBP1, producing the active transcription factor sXBP1, that upon translocation to the nucleus, leads to the expression of target genes, often involved in ERAD , 29. OurThe UPR response can be elicited by a variety of stimuli, including defects in protein folding or protein-folding overload in the ER. To investigate whether the observed differences after H6PD down-regulation in the UPR protein levels are a consequence of changes in protein folding, we analyzed the expression of members from the 3 main protein-folding families , 31. Fir2 were used . We observed a significant increase in FRET efficiency both at 42 and 72 h after H6PD knockdown, which translates into increased ER luminal calcium concentrations, starting at least 42 h after siRNA treatment . Unfortunately, we were not able to successfully implement the ER-specific hyper probe in our cell system . Given tPrevious work showed that H6PD down-regulation in 2 murine cancer cell lines decreased overall ATP levels, without affecting mitochondrial oxygen consumption. In addition, supernatant lactate concentration, indicative of glycolysis, was found to be decreased . In the 1 phase. G1 block after H6PD down-regulation has been reported before, albeit with a more pronounced difference   were decMice lacking H6PD present with skeletal muscle defects and muscles cells from these animals exhibited increased Grp78 protein expression, in addition to alterations in different UPR proteins, as shown by microarrays, including an induction of CHOP . Using tCHOP and XBP1 expression   , 50 and and P62) , 52. Furand P62) , 54.The reliance of cancer cells on the UPR pathway has been well established, and increased levels of critical UPR components have been shown in a multitude of human cancer tissues, including those originating in breast, colon, lung, pancreas, skin, prostate, and brain , 55, 56.57In an attempt to elucidate the mechanism underlying the attenuation of UPR after H6PD silencing, we examined a potential involvement of impaired protein folding. We found significantly increased levels of calreticulin, a calcium-binding protein that surveils proper folding of glycoproteins in the ER lumen before exit to the Golgi . In addiAccording to a previous study, knockdown ofH6PDin 2 murine cancer cell lines caused a reduction in ATP content, which was attributed to a reduction in glucose consumption, whereas no difference in oxidative phosphorylationwas observed (9). In the present study, in analyzing SUM159 cells, we did not find differences in totalATP levels afterH6PDdown-regulation.As different cancer cells can display profound differences in their metabolic capacity and in using different energy sources, we determinedthe impact ofH6PDknockdown on energy metabolism in the 2 breast cancer cell lines SUM159 and MDA-MB-453, both in their basal state and under stressed conditions by treatment with the mitochondrial electron transport chain inhibitor antimycin and the oxidative phosphorylation uncoupler FCCP. Comparison of the baseline and stressed phenotype provides insight into the metabolic potential of each cell line. Knockdown of H6PD led to increased ECAR in the SUM159 cells and decreased OCR in the MDA-MB-453 cells at steady state. Analysis of the rate of OCR/ECAR utilization D showed The results presented highlight the importance of NADPH production in the ER through H6PD for cancer cells. We demonstrated a significant role for H6PD in promoting cell proliferation and migration. Moreover, we described the cellular changes after H6PD silencing that could underpin these effects, including an increased ER luminal oxidation state within the ER, an attenuation of the UPR pathway and an increase in ER calcium concentrations. The ER is a dynamic organelle where vital functions take place, encompassing, among others, protein synthesis and quality control, lipid synthesis, and calcium signaling. Our study opens new avenues into understanding how compromised ER homeostasis influences cancer cell physiology and proposes H6PD as a potential novel therapeutic target against cancer.http://www.fasebj.org to obtain this information.This article includes supplemental data. 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Mammalian patatin-like phospholipase domain–containing proteins (PNPLAs) are lipid-metabolizing enzymes with essential roles in energy metabolism, skin barrier development, and brain function. A detailed annotation of enzymatic activities and structure–function relationships remains an important prerequisite to understand PNPLA functions in (patho-)physiology, for example, in disorders such as neutral lipid storage disease, non-alcoholic fatty liver disease, and neurodegenerative syndromes. In this study, we characterized the structural features controlling the subcellular localization and enzymatic activity of PNPLA7, a poorly annotated phospholipase linked to insulin signaling and energy metabolism. We show that PNPLA7 is an endoplasmic reticulum (ER) transmembrane protein that specifically promotes hydrolysis of lysophosphatidylcholine in mammalian cells. We found that transmembrane and regulatory domains in the PNPLA7 N-terminal region cooperate to regulate ER targeting but are dispensable for substrate hydrolysis. Enzymatic activity is instead mediated by the C-terminal domain, which maintains full catalytic competence even in the absence of N-terminal regions. Upon elevated fatty acid flux, the catalytic domain targets cellular lipid droplets and promotes interactions of PNPLA7 with these organelles in response to increased cAMP levels. We conclude that PNPLA7 acts as an ER-anchored lysophosphatidylcholine hydrolase that is composed of specific functional domains mediating catalytic activity, subcellular positioning, and interactions with cellular organelles. Our study provides critical structural insights into an evolutionarily conserved class of phospholipid-metabolizing enzymes. PNPLA genes including neutral lipid storage disease, non-alcoholic fatty liver disease, ichthyosis, hereditary spastic paraplegia, and other neurodegenerative syndromes 1–6–10–yndromes , 13, 14.yndromes , 16.PNPLA6 gene have been linked to a complex spectrum of neuroendocrine disorders including ataxia, spastic paraplegia, chorioretinopathy, and hypopituitarism  of the closely related PNPLA7 . Both proteins share a highly conserved domain architecture that is assembled of the enzymatic patatin-like phospholipase domain and extensive “non-enzymatic” segments of poorly defined function including three putative cyclic nucleotide monophosphate (cNMP)-binding sites , 25. Initrations . In mammtrations . AlthougIn this study, we further characterized the enzymatic function of PNPLA7 in cellular lipid metabolism and established detailed structure–function relationships among domain architecture, subcellular positioning, and enzymatic activity of the protein. We confirm that PNPLA7 acts as a potent intracellular lysophospholipase and identify LPC as a major substrate of PNPLA7 in living cells. Moreover, we demonstrate that PNPLA7 is composed of specific functional parts mediating ER targeting, interactions with LDs, and substrate hydrolysis. Our study provides novel structural insights into an evolutionarily conserved class of phospholipid-metabolizing enzymes.A). The lipid hydrolase activity of PNPLA7-EGFP was 2.6-fold higher toward C18:1 LPC as compared with C18:0 LPC, suggesting a preference of the enzyme for unsaturated compared with saturated species . Lipid analysis revealed that stable expression of PNPLA7-EGFP significantly reduced cellular LPC levels (−34%) as compared with EGFP-expressing control cells but did not affect total cellular levels of LPE, LPS, PC, PE, or PS . The decrease in LPC levels observed in PNPLA7-EGFP-expressing cells was mainly due to a reduction in unsaturated LPC species containing C16:1 (−35%), C18:1 (−43%), and C18:2 (−52%) fatty acids (FAs) . Although a trend toward decreased cellular levels was also observed for other LPC species, the difference was not significant when compared with controls . To further investigate the function of PNPLA7 in cellular LPC metabolism, we depleted endogenous PNPLA7 in murine AML12 cells by RNAi and assessed cellular LPC hydrolase activities and LPC levels. Stable expression of two independent shRNA constructs targeting PNPLA7 (shRNA1 and shRNA2) decreased cellular Pnpla7 mRNA concentrations by 59 and 63%, respectively, as compared with cells expressing scrambled shRNA (A). This was accompanied by decreased PNPLA7 protein levels in cells expressing either shRNA1 or shRNA2, whereas expression of the closely related PNPLA6 was unchanged (B). The reduction in PNPLA7 expression was associated with a 16–20% decrease in cellular LPC hydrolase activity  but did not affect total cellular LPC levels . Taken together, these data provide evidence that PNPLA7 acts as an intracellular LPC-preferring lysophospholipase that contributes to cellular LPC hydrolase activity in mammalian cells.To assess possible function(s) of PNPLA7 in cellular lipid metabolism, we first created cell lines stably expressing PNPLA7-EGFP or EGFP, respectively, and subjected them to enzyme activity assays and lipid analysis. Consistent with previous studies, cell homogenates expressing PNPLA7-EGFP exhibited increased hydrolytic activity toward several lysophospholipid species as compared with EGFP-expressing control homogenates including C18:1 LPC (4.6-fold), C18:1 lysophosphatidylethanolamine , and C18:1 lysophosphatidylserine  . In addiFig. 1ed shRNA A. This wnchanged B. The reA. The N-terminal half of the protein includes a single predicted transmembrane (TM) domain close to the N terminus and a putative “regulatory” (R) domain, which harbors three predicted cNMP-binding domains (CBDs). The C-terminal half (C domain) includes the patatin-like phospholipase domain predicted to mediate enzymatic activity . Subcellular fractionation of Neuro-2a postnuclear supernatants (PNSs) and subsequent immunoblotting showed that endogenous PNPLA7 was enriched in the membrane fraction but undetectable in the cytosol . Likewise, a PNPLA7-EGFP fusion protein transiently expressed in COS-7 cells was exclusively recovered in the membrane fraction , indicating a similar subcellular distribution of endogenous PNPLA7 and ectopically expressed PNPLA7-EGFP. Membrane-associated PNPLA7-EGFP was partially solubilized with Triton X-100 but not with NaCl or Na2CO3, similar to the integral membrane protein Calnexin but unlike the peripheral membrane protein DDHD2 . Next, we used proteinase K protection assays to assess the topological orientation of PNPLA7 within cellular membranes. Incubation of isolated microsomes with increasing concentrations of proteinase K provoked a dose-dependent depletion of the signal corresponding to full-length PNPLA7-EGFP in our immunoblots . This was accompanied by the generation of a ∼25-kDa fragment likely representing EGFP, which is known to be resistant to proteinase K digestion, indicating proteolytic degradation and therefore cytosolic orientation of the C terminus of PNPLA7 . Using antibodies directed toward the C domain or the R domain of PNPLA7, we failed to detect additional protein bands eluding proteolytic digestion of PNPLA7-EGFP, suggesting that these domains are also largely exposed to the cytosol and thus accessible to proteolytic degradation . Integrity of the isolated microsomes was confirmed by the apparent protection of the 66-kDa luminal domain of Calnexin from proteolytic degradation . To address whether the N terminus of PNPLA7 is oriented toward the cytosol or the lumen, we repeated the proteinase protection assay with microsomes isolated from COS-7 cells expressing PNPLA7 harboring an N-terminal HA tag (HA-PNPLA7). As shown in Fig. 3E, proteinase K treatment depleted the signal corresponding to full-length HA-PNPLA7 and led to the accumulation of an immunoreactive peptide of less than 10 kDa consistent with a luminal orientation of the N terminus. Addition of Triton X-100 to solubilize microsomal membranes rendered this peptide susceptible to proteolytic digestion, suggesting that microsomal integrity is indeed required to protect the N-terminal part of PNPLA7 from proteolysis. In an independent approach, we permeabilized COS-7 cells expressing HA-PNPLA7 either with Triton X-100 or digitonin and costained the cells with antibodies directed toward the HA tag and the C domain . Permeabilization with Triton X-100 rendered both epitopes accessible to antibody staining, resulting in a reticular staining pattern reminiscent of the ER and an apparent overlap of both signals. In contrast, digitonin treatment, which is used to selectively permeabilize the plasma membrane, rendered only the C region but not the N-terminal HA tag accessible to antibody staining . Taken together these results suggest that PNPLA7 is a bilayer-spanning TM protein that orients its N terminus toward the lumen but exposes its R and C domains largely toward the cytosol.To link the lipid-metabolizing function of PNPLA7 to a specific cellular site, we next assessed its subcellular localization and the topological orientation of its domains. The arrangement of possible functional domains within the PNPLA7 polypeptide is shown in Fig. 3in DDHD2 , 29  and assessed their subcellular localization in living COS-7 cells by confocal fluorescence microscopy. Consistent with the results described above, ectopically expressed full-length PNPLA7-EGFP exhibited a reticulate pattern that colocalized with an ER marker protein in COS-7 cells (B). A PNPLA7 mutant lacking the C domain (PNPLA7-N-EGFP) exhibited a similar subcellular distribution as full-length PNPLA7-EGFP and colocalized with the ER, indicating that the C domain is dispensable for ER association of PNPLA7. Furthermore, sole expression of the N-terminal TM domain (PNPLA7-TM-EGFP) was sufficient to target EGFP to the ER, whereas deletion of the TM domain (PNPLA7-ΔTM-EGFP) compromised ER localization of PNPLA7, suggesting an essential requirement of the TM domain for ER targeting of PNPLA7. Notably, expression of a construct harboring an internal deletion of the R but not the TM domain resulted in aggregation of the recombinant protein that failed to properly distribute to the ER network. This suggests that both the TM domain and the R domain contribute to the positioning of full-length PNPLA7 at the ER. Consistent with these findings, a mutant lacking both the TM and R domains (PNPLA7-C-EGFP) distributed diffusely in the cytoplasm and showed only minimal overlap with the ER marker (B). As a complementary approach, we used subcellular fractionation and immunoblotting to assess membrane association of PNPLA7 mutant proteins. As shown in Fig. 4C, PNPLA7-EGFP, PNPLA7-N-EGFP, PNPLA7-TM-EGFP, and PNPLA7-ΔR-EGFP were recovered exclusively in the membrane fraction, whereas PNPLA7-ΔTM-EGFP and PNPLA7-C-EGFP distributed between the soluble and particulate fractions, suggesting impaired membrane association of these mutants. Thus, our data suggest that the N-terminal TM domain mediates efficient membrane tethering of PNPLA7 and that both the TM and R domains but not the C domain control the subcellular positioning of PNPLA7 at the ER.We next addressed the functional contribution of individual protein domains to the subcellular positioning of PNPLA7. To this end, we constructed EGFP-tagged PNPLA7 variants lacking individual domains A and ass-7 cells B. A PNPLR marker B. As a cA). Likewise, neither PNPLA7-N-EGFP nor PNPLA7-ΔTM-EGFP exhibited apparent colocalization with LDs under these conditions. In contrast, PNPLA7-ΔR-EGFP showed punctate and semi-ringlike enrichments in close proximity to LDs, indicating a possible interaction of this truncation mutant with LDs. This phenomenon was even more pronounced for PNPLA7-C-EGFP, which presented as numerous ring-shaped structures surrounding cellular LDs . Moreover, PNPLA7-C-EGFP colocalized with ectopically expressed PLIN2-mCherry, a bona fide LD protein, suggesting that it indeed associates with the LD surface .In addition to the ER, PNPLA7 has been shown to localize to a subset of cellular LDs upon incubation of cells with FAs . To addrFig. 5 surface , 31 . Low levels of PNPLA7-EGFP, PNPLA7-ΔTM-EGFP, and PNPLA7-ΔR-EGFP were also detected in the LD fraction. However, the abundance of these proteins was much lower as compared with PNPLA7-C-EGFP.To further study the interactions of PNPLA7 protein domains with LDs, we fractionated transfected COS-7 cells by ultracentrifugation and assessed the abundance of each PNPLA7 variant in the LD fraction by immunoblotting. Consistent with the microscopy data, ectopically expressed PNPLA7-C-EGFP was markedly enriched in the LD fraction, resembling the distribution of endogenous PLIN2 C. Low leA. Mutants containing truncations at the C terminus retained the ability to localize to LDs, suggesting that the patatin-like phospholipase domain is largely dispensable for LD targeting. In contrast, even minor truncations at the N terminus of the C domain compromised LD targeting. As shown in A and B, a stretch between amino acids 681 and 967 was sufficient for LD targeting, whereas further N- or C-terminal truncations abolished LD association of the C domain. Thus, a stretch of 287 amino acids is essential for the interactions between the C domain of PNPLA7 and LDs.To identify the minimal domain required for LD targeting of PNPLA7-C-EGFP, we further truncated the protein and assessed the ability of these mutants to colocalize with LDs. Schematic drawings of the truncations and their effects on LD targeting are summarized in Fig. 6A), we next asked whether the presence of cNMP analogs would affect the subcellular distribution of PNPLA7. Incubation of cells with 8-CPT-cAMP provoked a subcellular redistribution of ectopically expressed PNPLA7-EGFP, which manifested as ring- and semi-ringlike enrichments of the protein close to the LD surface on top of its reticulate ER-like distribution . A similar response was elicited by 8-CPT-cGMP (data not shown). In contrast to PNPLA7-EGFP, PNPLA7-N-EGFP was not enriched at LDs under identical conditions. Furthermore, deletion of CBD3 but not CBD1 or CBD2 abolished LD interactions of PNPLA7-EGFP (A and B). In summary, these data show that the C domain localizes to cellular LDs and suggest an involvement of both the C domain and CBD3 in LD interactions of PNPLA7 in response to elevated cNMP levels. Noteworthy, the specific C18:1 LPC hydrolase activity of PNPLA7 was largely unchanged upon incubation of cells with FAs and/or cNMPs (C) or starvation (data not shown), suggesting that alterations in subcellular distribution do not alter PNPLA7 activity.Because the N-terminal region contains three putative CBDs A, we nexribution B, inset.LA7-EGFP , A and Bor cNMPs C or starA). As shown in Fig. 8B, ectopic expression of PNPLA7-EGFP or PNPLA7-C-EGFP increased cellular LPC hydrolase activity to a similar extent (2.1–2.3-fold) as compared with EGFP-expressing controls, suggesting that the C domain maintains full catalytic competence also in the absence of TM and R domains. The specific activities of PNPLA7-ΔTM-EGFP and PNPLA7-ΔR-EGFP were 3.0- and 3.3-fold higher than the activity of EGFP-expressing controls, thereby moderately exceeding the activity of PNPLA7-EGFP. In contrast, expression of PNPLA7-N-EGFP failed to increase cellular lysophospholipase activity, which is consistent with a requirement of the C domain for substrate hydrolysis . To assess whether the catalytic C domain is functional also in intact cells, we generated COS-7 lines stably expressing PNPLA7-C-EGFP or EGFP, respectively, and performed lipid analyses. Expression of PNPLA7-C-EGFP reduced cellular levels of LPC by 42% compared with EGFP-expressing control cells but did not affect cellular levels of PC, PE, PS, LPE, and LPS . Thus, the C domain is both required and sufficient to mediate catalytic activity of PNPLA7 in mammalian cells.We finally aimed to understand how specific protein domains regulate the enzymatic function of PNPLA7. To do so, we expressed PNPLA7-EGFP or truncated PNPLA7-EGFP mutant proteins in COS-7 cells and compared cellular lysophospholipase activities. Immunoblotting revealed similar expression levels of PNPLA7-EGFP and truncated versions of the protein A. As shoin vivo  and/or a specific subset of LDs (e.g. ER-associated or “nascent” LDs).We initially described PNPLA7 as a lipid hydrolase with a dual subcellular localization at the ER and LDs . Our newAdditional structure–function analyses revealed that the C domain constitutes the major catalytic part of PNPLA7 and thus serves a dual role in subcellular localization and substrate hydrolysis. Expression of full-length PNPLA7 or the isolated C domain increased cellular lysophospholipase activity to a similar extent, suggesting that N-terminal domains neither restrict nor promote catalytic activity of PNPLA7. Consistent with this finding, it has been shown that the C-terminal esterase domain of the homologous human protein PNPLA6 acquires catalytic competence also in the absence of N-terminal regions . Sole exsn-glycero-3-phosphocholine), C16:0 LPC , C18:1 LPC (1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine), C18:1 LPS (1-oleoyl-2-hydroxy-sn-glycero-3-phospho-l-serine), C18:1 LPE (1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine), C18:1/C18:1 PC , C18:1/C18:1 PE , and C18:1/C18:1 PS  were obtained from Avanti Polar Lipids . l-α-Lysophosphatidylcholine (ART1809) was obtained from American Radiolabeled Chemicals . HCS LipidTOXTM Deep Red was obtained from Thermo Fisher Scientific . 8-CPT-cAMP was purchased from Abcam , and 8-CPT-cGMP was purchased from Sigma-Aldrich.C18:0 LPC (1-stearyl-2-hydroxy-TM sheep anti-mouse IgG antibody (NA931) linked to horseradish peroxidase (HRP) was purchased from GE Healthcare. An HRP-linked goat anti-rabbit IgG antibody (PI-1000) was purchased from Vector Laboratories . An HRP-linked goat anti-guinea pig IgG antibody (6090-04) was purchased from Southern Biotechnology . A Rhodamine RedTM-X-linked goat anti-mouse IgG antibody was obtained from Jackson ImmunoResearch Laboratories , and DyLight® 488-conjugated goat anti-rabbit IgG antibody was obtained from Thermo Fisher Scientific.A rabbit anti-GFP antibody (ab6556) was purchased from Abcam. A rabbit anti-PNPLA7 antibody (HPA009130), which is directed to the C domain of PNPLA7, and a mouse anti-HA antibody (H3663) were obtained from Sigma-Aldrich. A guinea pig anti-PLIN2 antibody (GP40) was obtained from PROGEN . A rabbit anti-DDHD2 antibody (25203-1-AP) was obtained from Proteintech . Rabbit anti-Calnexin (2679), rabbit anti-GAPDH (2118), and rabbit anti-IRE1α (3294) antibodies were obtained from Cell Signaling Technology . Antisera against the R domains of murine PNPLA7 and PNPLA6 were generated by repetitive immunizations of rabbits with the peptides CSVPLPSNHGEVDELRQSQGSGSNT and PAGDPVKPTSLEAPPAPLLSRC, respectively, which were coupled to keyhole limpet hemocyanin as carrier . An ECLProtein domains were analyzed using the protein family database (Pfam), an online database containing collections of protein domains and families . TM domaThe vectors pDsRed2-ER, pmCherry-N1, and pEGFP-N3 were obtained from Clontech. pEGFP-N1 containing the coding sequence (cds) of human PLIN2 was a kind gift from Stefan Höning . To generate a plasmid encoding PLIN2-mCherry, the cds of PLIN2 was amplified using the primers 5′-GCG AAT TCG CCA TGG CAT CCG TTG CAG TTG A-3′ and 5′-CAA CCG GTC GAT GAG TTT TAT GCT CAG ATC G-3′, and the PCR product was digested with EcoRI and AgeI and ligated into the multiple cloning site of pmCherry-N1. The cds of PNPLA7 was amplified by PCR using cDNA of murine cardiac muscle as template and the primers 5′-TTC TCG AGG CCA TGG AGG AGC AGT CCC AGT CC-3′ (F1) and 5′-GCG AAT TCG ACG AAG GAT GTT CCA GTC TTG G-3′ (R1). PNPLA7 truncation mutants were generated by PCR using the primers 5′-TTC TCG AGG CCA TGG CAG AAC CCA CTC CTC AGT AC-3′ (F2) and R1 for ΔTM-PNPLA7 (Δ1–40), 5′-TTC TCG AGG CCA TGG TTG TAA CGC GAC TGA TTC ATC TC-3′ (F3) and R1 for PNPLA7-C (Δ1–680), 5′-GCG AAT TCG CCA TGG AGG AGC AGT CCC AGT C-3′ (F4) and 5′-CAG GAT CCC CGT CGT AAT CTT CTC ACT C-3′ (R2) for TM-PNPLA7 (Δ41–1326), F1 and 5′-GCG AAT TCG ACT GTG GGT ACC TGC GCT TGA TAG-3′ (R3) for PNPLA7-N (Δ681–1326), F3 and 5′-GCG AAT TCG ATC TTG CCA CAT CCG CTG GGA G-3′ (R4) for PNPLA7-C1 , F3 and 5′-GCG AAT TCG AGA AGA TGT TGC TGA TGC TGC-3′ (R5) for PNPLA7-C2 , F3 and 5′-GCG AAT TCG AAG CAA ACA GAG CAC CCA TGA A-3′ (R6) for PNPLA7-C3 , F3 and 5′-GCG AAT TCG AGG CAC ACC CTC TAG CTC CAC C-3′ (R7) for PNPLA7-C4 , 5′-TTC TCG AGG CCA TGT TTG CTC TGG AGC TCC AAC A-3′ (F4) and R1 for PNPLA7-C5 (Δ1–741), 5′-TTC TCG AGG CCA TGT TGG TGC TTG GAG GGG GTG GA-3′ (F5) and R1 for PNPLA7-C6 (Δ1–923), 5′-TTC TCG AGG CCA TGT CCA TGG GGG CAA AGG TTG TG-3′ (F6) and R1 for PNPLA7-C7 (Δ1–1090), and F4 and R6 for PNPLA7-C8 . The PCR products were purified by agarose gel electrophoresis and inserted into the multiple cloning site of pEGFP-N3 using the XhoI and EcoRI restriction sites to create C-terminal in-frame fusions with EGFP. Internal deletion mutants of PNPLA7 were generated using the Q5® site-directed mutagenesis kit with PNPLA7-EGFP as template and the primers 5′-CCG TCG TAA TCT TCT CAC-3′ and 5′-GTT GTA ACG CGA CTG ATT C-3′ for ΔR-PNPLA7 (Δ41–680), 5′-CAG ATC ATC ATG GTC CGG-3′ and 5′-CCG AAC ATT TTT CAA CAT GTA C-3′ for ΔCBD1 (Δ144–265), 5′-GTC CTG GGC GTG GCA CAC-3′ and 5′-GTC CTT TGT GGC AGC TCT GAA G-3′ for ΔCBD2 (Δ455–565), and 5′-GTT GTA ACG CGA CTG ATT C-3′ and 5′-CAT CCT CTT CAC AAC TGT G-3′ for ΔCBD3 (Δ578–680). A DNA fragment encoding for PNPLA7 harboring an N-terminal HA tag was created by PCR using the primers 5′-TAA TCT CGA GGC CAC CAT GTA CCC ATA CGA TGT TCC AGA TTA CGC TAT GGA GGA GCA GTC CCA GTC-3′ and 5′-GAC GAA TTC GTC AGG AAG GAT GTT CCA GTC-3′. The PCR product was purified as described above and inserted into the multiple cloning site of pEGFP-N3 using the XhoI and EcoRI restriction sites. To generate lentiviral expression vectors, pEGFP-N3 constructs encoding the open reading frames for EGFP, PNPLA7-EGFP, and PNPLA7-C-EGFP were digested with XhoI and NotI, and the resulting fragments were inserted into the multiple cloning site of pLVX IRES Puro (Clontech). Mission® lentiviral pLKO.1 vectors encoding for scrambled shRNA or shRNAs targeting murine PNPLA7 were obtained from Sigma-Aldrich. Constructs used for RNAi experiments targeted the sequences CCAAGAGGATTCTGCGCTTTA (shRNA1) and CATGTCCTTGTCAGGCTATAT (shRNA2) of the PNPLA7 cds.ml-glutamine, 1 mm sodium pyruvate, non-essential amino acids, 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin. COS-7 cells (ATCC CRL-1651) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. AML12 cells (ATCC CRL-2254) were cultured in a 1:1 mixture of DMEM and Ham's F-12 medium supplemented with 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 5 ng/ml selenium, and 40 ng/ml dexamethasone. All cells were maintained at 37 °C, 95% humidity, and 5% CO2. Cells were transfected with Metafectene®  according to the manufacturer's instructions and used for experiments 24 h thereafter. Cells were treated with 400 μm oleic acid bound to bovine serum albumin , 1 mm 8-CPT-cAMP, or 1 mm 8-CPT-cGMP for 16 h or with Hanks' buffered salt solution for 4 h before being processed for imaging or lipid hydrolase assays.Neuro-2a cells (ATCC CCL-131) were maintained in minimum essential medium supplemented with 2 mLentiviral particles harboring pLVX IRES Puro vectors for the expression of EGFP-tagged proteins or pLKO.1 vectors for the expression of shRNAs were generated in HEK293T cells (ATCC CRL-3216) according to the manufacturer's instructions (Clontech). Before transduction, COS-7 or AML12 cells were seeded into 6-well plates at a density of 300,000 cells/well. Cells were incubated for 24 h with lentivirus-containing supernatants in the presence of 8 μg/ml Polybrene. To select for stable expression, cells were maintained for 7 days in medium containing 2 μg/ml puromycin.6 cells) were extracted twice according to Folch et al.  for UPLC-triple quadrupole analysis. Chromatographic separation was modified after Knittelfelder et al.  starting with a 25-min gradient with 100% solvent A , 10 mm ammonium acetate, 0.1% formic acid). An EVOQ EliteTM triple quadrupole mass spectrometer (Bruker) equipped with an electrospray ionization source was used for detection. Lipid species were analyzed by selected reaction monitoring . Data acquisition was done by MS Workstation (Bruker). Data were normalized for recovery and extraction and for ionization efficacy by calculating analyte/internal standard ratios.Total lipids of cell pellets , by passing 30 times through a 26-gauge needle on ice. PNSs were obtained by centrifugation at 4 °C and 1,000 × g for 10 min. PNS was further fractionated into cytosol and membrane fractions by ultracentrifugation at 4 °C and 100,000 × g for 60 min. For the isolation of lipid droplets, cells were homogenized on ice with a Dounce homogenizer in 20 mm Tris/HCl, 1 mm EDTA, pH 7.4, and lipid droplets were isolated by ultracentrifugation using a discontinuous sucrose gradient according to Brasaemle and Wolins (TM Protein Assay kit according to the manufacturer's protocol (Thermo Scientific).COS-7 cells were washed with phosphate-buffered saline (PBS), scraped from tissue culture plates, and collected by brief centrifugation. For the preparation of membrane and cytosol, cells were homogenized in 10 md Wolins  with minm NaCl, 0.1 m Na2CO3, or 1% Triton X-100 for 30 min on ice. Afterward, soluble and insoluble components were separated by centrifugation at 4 °C and 100,000 × g for 30 min and subjected to immunoblotting. For membrane protection assays, isolated membrane fractions were incubated in the presence of 0–10 μg/ml proteinase K in the absence or presence of 1% Triton X-100 for 15 min at room temperature. The reactions were stopped by the addition of PMSF at a final concentration of 2 mm, further incubated for 5 min, and subjected to immunoblotting.Membrane fractions of COS-7 cells were resuspended in buffer A and incubated in the presence of 1 m Tris/HCl, 150 mm NaCl, 0.1% Tween 20, pH 7.5 (TBST), containing 10% milk to block unspecific binding sites. Primary and secondary antibodies were diluted in TBST containing 5% milk and incubated with the membrane for 1 h at room temperature followed by extensive washing with TBST.Samples were mixed with Laemmli buffer, denatured for 5 min, subjected to SDS-PAGE, and electroblotted onto PVDF membranes. Membranes were incubated for 1 h with 20 mTM, Life Technologies) according to the manufacturer's instructions. RT-qPCR was performed as described previously  and the MaximaTM SYBR Green/ROX PCR reaction mixture . The following primers were used: Pnpla7 fw, 5′-CGT GTT TTC CAA CGA CCA CC-3′; Pnpla7 rv, 5′-TCT GCT AGT GCC CTG AGG AT-3′; 36B4 fw, 5′-GCT TCA TTG TGG GAG CAG ACA-3′; 36B4 rv, 5′-CAT GGT GTT CTT GCC CAT CAG-3′. Relative mRNA levels were quantified according to the ΔΔCt method using 36B4 as reference gene.Cells were harvested and snap frozen in liquid nitrogen. RNA was extracted using the TRIzol® reagent  followed by centrifugation at 4 °C and 1,000 × g. Lipid substrates were prepared by sonication of glycerophospholipids in 200 mm Bistris propane buffer, pH 7, containing 10 mm CHAPS, 1 mm EDTA, and 600 mm NaCl. Unless otherwise indicated, substrates contained lysophospholipids at final concentrations of 3 mm or diacylglycerophospholipids at final concentrations of 1 mm. Assays were started by the addition of 50 μl of substrate to 50-μl samples in solution A and incubated under steady shaking at 37 °C in a water bath. Substrate hydrolysis is expressed as release of FAs, which was determined routinely using a commercially available kit (non-esterified FA (NEFA) reagent, Wako, Neuss, Germany) according to the manufacturer's instructions. For the determination of endogenous LPC hydrolase activities in AML12 cells, 3H-labeled LPC at a specific activity of 0.33 μCi/μmol was included in the substrate. After incubation, assays were extracted with 0.5 ml of chloroform/methanol/acetic acid , and extracts were separated by thin layer chromatography using Silica G plates and hexane/diethyl ether/acetic acid  as solvent. FAs were identified by comigrating authentic standards, and the associated radioactivity was determined by liquid scintillation counting. All assays were performed in triplicates.Protein samples for lipid hydrolase assays were prepared by sonication of COS-7 or AML12 cells in 0.25 COS-7 cells were seeded in chambers mounted onto coverslips  and transfected as described above. Cells expressing EGFP-tagged proteins were fixed with 4% paraformaldehyde for 20 min at room temperature, and LDs were counterstained using HCS LipidTOX Deep Red. Cells expressing HA-PNPLA7 were fixed with 4% paraformaldehyde for 20 min at room temperature and permeabilized for 15 min with PBS containing either 0.1% Triton X-100 or 0.0005% digitonin. Cells were washed three times with PBS and incubated at room temperature with PBS containing 1% BSA for 1 h to block unspecific binding. Primary antibodies were diluted in blocking solution and incubated with cells overnight at 4 °C. Cells were washed with PBS and incubated with secondary antibodies diluted in blocking solution at room temperature for 1 h followed by extensive washing with PBS. Cells were imaged using a Leica SP5 confocal microscope equipped with a Leica HCX 63× 1.4 numerical aperture oil immersion objective. EGFP and DyLight 488-conjugated antibodies were excited at 488 nm, and emission was detected between 500 and 530 nm. DsRed2, mCherry, and Rhodamine Red were excited at 561 nm, and emission was detected between 580 and 610 nm. HCS LipidTOX Deep Red was excited at 633 nm, and emission was detected between 650 and 700 nm.t test. Group differences were considered statistically significant for p < 0.05 (*).All measurements were performed in triplicates. Data are presented as means ± S.D. Statistical significance was determined by the Student's unpaired C. H., P.-A. C., and R. Z. conceived and coordinated the study and wrote the paper. H. X. and F. H. performed molecular biology work. C. H., B. K., and P.-A. C. performed all cellular, biochemical, and imaging experiments. T. O. E. performed lipid analyses. All authors reviewed the results and approved the final version of the manuscript.


Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (sleeping sickness), yet little is known about which PKs are essential for survival in mammals. A recent kinome-wide RNAi screen with 176 individual bloodstream form Trypanosoma brucei lines identified PKs required for proliferation in culture. In order to assess which PKs are also potential virulence factors essential in vivo, lines were pooled, inoculated into mice, and screened for loss of fitness after 48 h RNAi. The presence of trypanosomes in the bloodstream was assessed using RNAi target sequencing (RITseq) and compared to growth in culture. We identified 49 PKs with a significant loss of fitness in vivo in two independent experiments, and a strong correlation between in vitro and in vivo loss of fitness for the majority. Nine PKs had a more pronounced growth defect in vivo, than in vitro. Amongst these PKs were several with putative functions related to stress responses mediated through the PI3K/TOR or MAPK signaling cascades, which act to protect the parasite from complement-mediated and osmotic lysis. Identification of these virulence-associated PKs provides new insights into T. brucei-host interaction and reveals novel potential protein kinase drug targets.Protein kinases (PKs) are a class of druggable targets in  Trypanosoma brucei is an obligate parasite in the class kinetoplastida, a group of flagellated protozoa characterized by a dense complex of circular mitochondrial DNA called the kinetoplast. T. brucei spp infect humans and animals in sub-Saharan Africa, causing diseases that remain major threats to health and economies in the region. T. brucei exhibits a complex life cycle that requires adaptation to many different environments, including different compartments within the blood-feeding insect vector responsible for transmission between different mammalian hosts: the tsetse fly. Once in the mammal, T. brucei resides extracellularly in the bloodstream, tissue fluids, central nervous system and adipose tissue1. Bloodstream T. brucei undergo antigenic variation and express a single Variant Surface Glycoprotein (VSG) per cell, which can be switched upon expansion of the population to create diversity2. The host builds an adaptive immune response against at least the most abundant variants, leading to their clearance and enabling outgrowth of cells that have switched to an antigenically distinct VSG. Iteration of this process leads to the characteristic waves of parasitemia3.T. brucei protein kinome represents 2% of the parasite’s protein-coding capacity and comprises 157 conserved eukaryotic PKs (ePKs), 12 non-catalytic pseudokinases and 20 atypical PKs (aPKs)6. Substantial differences exist between the T. brucei and the human protein kinomes, as the parasites lack receptor-linked tyrosine kinases and tyrosine-like kinases. Despite this, tyrosine phosphorylation has been reported, possibly due to dual-specificity kinases5. T. brucei also has a relatively reduced representation of AGC and CAMK families, while CMGCs, STEs and NEKs are comparatively expanded. In addition, several highly divergent PKs are likely to play parasite-specific functions that may present targets for selective inhibition by small molecules5.Protein kinases (PKs) are key signalling proteins in eukaryotes, playing critical roles as central regulators in many biological functions, as well as being validated drug targets. The 7. Several high-throughput screening campaigns with compound libraries have been published linking in vitro trypanocidal activity with PK inhibition10, though the specific PK target in each case is unknown. Over 40 T. brucei PKs have been shown to be essential for normal cell proliferation in vitro11. Many biological roles for PKs have been revealed in T. brucei, but they have been observed mainly in the artificial conditions of in vitro culture. In this paper we use a kinome-focused RNAi library in a 72 h mouse infection model to address a key question of both biological and pharmaceutical relevance: which PKs are required for survival of the parasite in the environment of the mammalian bloodstream?PKs are a promising source of druggable targets, with more than 100 inhibitors already in clinical trials and successful drugs in the market, such as the prototypical compound Imatinib® for chronic myeloid leukemiaT. brucei RNAi cell lines to identify PKs essential for proliferation of bloodstream form parasites in culture, cell cycle regulators and negative regulators of BSF to PCF differentiation6. In order to increase the capacity for screening the kinome RNAi library, we made a pool of the 177 available cell lines, which targeted 183 of the PKs (6 were double knockdowns)6. This pool allowed parallel phenotyping of the population in a single culture (in vitro) and in a single mouse (in vivo) through RNAi target sequencing  with a bootstrap analysis including 1000 repeats, which enabled calculation of the median and estimation of 95% confidence intervals for each gene (Data sets in vivo. When the 0.6 (induced/uninduced) threshold was applied to the in vitro data set it was determined to be discriminatory, showing a progressive increase of the number of targets identified with time  and are likely to be kinetoplastid-specific. All of the 49 PKs have orthologues in either Leishmania major or Trypanosoma cruzi or both, though with variable levels of sequence identity between the kinetoplastid parasites  and Tb927.10.10350 (STE11/Bck1p), experienced growth retardation in vivo from 48 h, indicating a loss of fitness. For three other genes, Tb927.2.2430 (Other/CK2A2), Tb927.7.960 (CMGC/SRPK1) and Tb927.10.14300 (STE11/MRK1), the loss of fitness after in vivo RNAi was even more pronounced, since a decrease in parasitemia or clearance was seen before 96 h  to fresh rat serum for 3 h (10% and 50% concentration) and analyzed relative survival of RNAi induced and uninduced cells when compared to controls that were not exposed to rat serum , which caused impaired cell growth in vitro, caused no serum sensitivity. In contrast, RNAi against Tb927.2.1820 (CAMKL), Tb927.3.3920 (AUK2) and Tb927.7.960 (CMGC/SRPK1) resulted in significant, concentration-dependent sensitivity to serum, manifest in survival rates 50% lower than the equivalent uninduced samples. To understand whether such sensitivity was due to complement-mediated lysis, these three cell lines -RNAi induced and uninduced- were further exposed to 50% serum complement-inactivated with 20 mM EDTA17. For Tb927.2.1820 (CAMKL) and Tb927.7.960 (CMGC/SRPK1), the EDTA treatment ablated the serum sensitivity, strongly suggesting that the RNAi phenotype was due to increased complement-mediated killing. AUK2, however, was more difficult to interpret as Tet− cells showed increased survival when EDTA was added.One of the most obvious and distinctive elements encountered by parasites during infection in mice when compared with rum Fig. . In addiin vivo than in vitro  have a significantly more pronounced loss of fitness phenotype tro Fig. ; these htro Fig. . To testols Fig. . RNAi agT. brucei to survive in the mammalian bloodstream. We modified whole-genome parallel phenotyping based on RITseq technology12 and applied it to a defined collection of individually designed and validated RNAi cell lines. This method was used to analyze in vitro and in vivo experiments to permit the identification of 49 essential PKs, including 9 PKs that appear more important for survival in the environment of the mammalian circulatory system than in culture. Bioinformatic assessment of each gene suggested these PKs are involved in parasite stress-response pathways. Validation of individual cell lines identified PKs that play roles in protecting the parasite from mammalian serum factors and osmotic stresses. Our findings expand the repertoire of essential and potentially druggable PKs in this pathogen and begin to explore the mechanisms by which the parasite survives the multiple stresses of life inside its host.In this study we aimed to define the cohort of protein kinases that are essential for in vivo experiments for several reasons. The coefficient of determination between the induced/uninduced read mapping ratios obtained between the two experiments was very good, especially when focused on those with a loss of fitness RNAi phenotype , elements involved in stress management are also ‘indirect’ or ‘contributory’ elements required for establishment of the infection19. A connection between stress resistance and virulence has also been found for many other infectious eukaryotic organisms24, including Leishmania26. Sequence similarity between eight of the T. brucei in vivo-specific PKs and kinases in other eukaryotes suggests diverse functions, including gene expression, alternative splicing, protein synthesis and translational regulation in response to extracellular signals, and stress adaptation linked with autophagosome formation  PKs were identified amongst the in vivo cohort. Tb927.10.14300 (STE11/MRK1) has features in common with members of the yeast High Osmolarity Glycerol (HOG) pathway, which responds to osmotic challenge through actin recovery29, while the MORN motifs-containing protein Tb927.10.10350 (STE11/Bck1p) resembles human MEKK3, a positive regulator of the stress-activated protein kinase (SAPK) pathway30 putatively involved in cell integrity maintenance under stress conditions. The increased susceptibility to osmotic shock seen after RNAi induction of these two STE11 PKs  were not identified in this screen, which may suggest redundancy amongst the T. brucei STE7 cohort, or perhaps a non-canonical mode of action for the T. brucei STE11 PKs. Two other MAPKs (ERK-like), often involved in transcriptional and non-transcriptional regulation in response to external stimuli, were found to be essential both in vivo and in vitro: KFR1, previously reported to be regulated by interferon γ in order to promote proliferation in T. brucei32; and MAPK6 (ERK8), which regulates cytokinesis33.Trypanosomes are highly adaptable organisms, capable of withstanding immune assault by the host and responding to the many changing conditions they experience during their life cycles, including temperature, pH, osmotic imbalance, shear-forces, and availability of carbon and nitrogen sources. This study reveals that depletion of at least nine PKs limits the capacity of Data set . One of PKs Fig.  indicatein vivo cluster together within the PI3K/TOR pathway: Tb927.11.9190 (VPS15), Tb927.11.850 (Bud32) and Tb927.7.960 (SRPK1). VPS15 is required for stress-induced and developmentally-triggered autophagosome formation in other eukaryotes and assembles within the PI3K complexes34. PI3K signalling in yeast involves Bud32 , which acts as a transducer for TOR activation that, in turn, has also been related with autophagy and endocytosis under stress conditions. SRPK1 regulates splicing factors in response to stress and can be activated via PI3K signaling in other eukaryotes35. The identification of these TOR-related PKs is of interest, because TOR pathways are central for parasite-host interaction in T. brucei36. While most eukaryotes encode two TOR paralogues, trypanosomatids are the only known organisms to contain four, each integrated in a different protein complex, of which three are required for normal fitness both in vivo and in vitro . The three showed an increased sensitivity to serum upon RNAi induction, related to complement-mediated lysis. In what way, if at all, the two phenotypes might overlap is unclear but the potentially common function of each T. brucei PK in providing serum resistance . The final gene that is only essential for in vivo growth, CK2A2, has validated protein kinase activity and localizes primarily to the nucleolus42. CK2 is mainly controlled by distribution and abundance, with stress-induced mobilization extensively reported43. CK2 has also been associated with autophagy through intersection with PI3K/AKT/TOR and MAPK pathways44.It is intriguing that three PKs with a pronounced loss of fitness nce Fig.  might guT. brucei protein kinases are druggable45 and this work provides genetic validation for 49 protein kinases that are required for in vivo fitness. A phylogenetic comparative analysis between the T. brucei and Homo sapiens kinomes showed that 13 out of the 49 PKs had close human orthologues targeted by at least one of the compounds in the ‘HAT box’  + 10% v/v foetal bovine serum (GIBCO), Pen/Strep solution ] at 37 °C/5% CO2 in vented flasks. Selective antibiotics were used as follows: 0.2 μg ml−1 puromycin, 0.5 μg ml−1 phleomycin (InvivoGen), 2.5 μg ml−1 hygromycin B . RNAi was induced in vitro with tetracycline (Sigma Aldrich) in 70% ethanol at 1 μg ml−1.Monomorphic 6 cells ml−1 were exposed to fresh rat serum for 3 h (10% and 50% concentration). Relative survival was measured of induced (for 48 h) and uninduced clones compared to no-serum controls. The whole experiment was performed in triplicate.To test for serum resistance 200 μl of log-phase parasites in HMI11 at 1018 2 × 106 cells were resuspended for 5 min at 4 °C in 0.5 ml of a solution 55 mM KCl/1 mM glucose to induce cell swelling. 0.5 ml of a 263 mM KCl/1.75 mM Mg2Cl shrinking solution was added on top and cells incubated for 5 min at 4 °C. Remaining cells were washed once and resuspended in 1 ml of HMI11 media before counting. Survival of the induced cells compared to uninduced controls was plotted and significant results (p < 0.05) after T-tests indicated with a star.To test for response to osmotic shock6. RNAi lines were pooled into 9 ‘Mixed Stabilate Trypanosome Libraries’ (MSTL 1–9), each containing 19–25 cell lines. Then MSTLs were further pooled to make the final culture.All RNAi cell lines in this work were generated as previously describedin vitro RITseq was defrosted, grown for 24 h and diluted to contain 1 × 105 cells ml−1 in 100 ml. In triplicate the culture was split into two 50 ml flasks (tetracycline induced and uninduced control) and grown for 120 h, reducing cells to 1 × 105 cells ml−1 every 24 h. 1 × 107 cells were sampled daily for analysis.The library used for the ad libitum. Infections were performed by intraperitoneal injection and were evaluated daily by assessing parasitemia levels with a hemocytometer by tail vein sampling diluting blood in anticoagulant (CBSS/Heparin) 0.83% w/v. If parasitemia exceeded 108 cells ml−1 mice were euthanized with carbon dioxide and cervical dislocation. RNAi was induced in mice with doxycycline hyclate (Sigma Aldrich) added in sugared drinking water (0.2 g/L plus 50 g/L sucrose). For the in vivo RITseq, blood from euthanized mice 72 h after infection was collected and immediately applied to DEAE-cellulose columns as described50. DNA was isolated from the parasites using a QIAamp DNA kit .Female CD1 outbred mice (6–8 weeks old) were obtained from Charles River . Animals were given water and food A-3′) was used to PCR enrich RNAi inserts amplified from 10 ng of genomic DNA obtained per sample in a 50 µl reaction using Q5® High-Fidelity DNA polymerase . The PCR program was: 3 minutes at 98 °C; followed by 28 cycles comprising: 10 seconds at 98 °C, 10 seconds at 61 °C and 30 seconds at 72 °C; with a final extension step at 72 °C for 10 minutes. PCR products were cleaned up with Minielute PCR purification kit , 400 ng of DNA per sample were fragmented, size selected (average read length after sequencing ~106 nucleotides) and processed according to Ion Torrent library prep protocols. The multiplexed sample was subjected to emulsion PCR and Ion Torrent sequencing. The library pool used at Experiment 2 was processed as above with the following exceptions, a mix of MSTLs 1–9, PCR annealing temperature was elevated to 64 °C, as additional 6 nucleotide-long labels were added to the single universal primers 51, using default parameters. For in vivo Exp.1, single end reads generated from each sample were selected by the presence of a 9 base diagnostic tag [GCCAACTTT] present within the universal primers, permitting 1 mismatch . Approximately 20% of the reads contained the RNAi cassette. For in vivo Exp.2 and in vitro RITseq, single end reads were identified and clustered according to 6 base-long barcodes before being processed as in Exp.1 (without the fragmentation/size selection step) finding 65–70% of reads containing the RNAi target. Selected reads were mapped to the artificial chromosomes with Bowtie2 . The “.sam” format files generated by Bowtie2 were parsed and the coordinates of the appropriate chromosome to which the read mapped were recorded. The mapped reads were assigned to the appropriate PK sequence by reference to the index generated above. The read was assigned if it lay entirely within the PK sequence or overlapped the 3′ or 5′ end of the PK sequence. In each replicate, accumulated read abundances were normalized by multiplying raw counts 106 times, dividing by the sum of total valid reads accepted for the analysis in the whole sample and rounding to the next integer, i. e. reads per million (RPM). Bowtie2 mapping was used also to compare whole-genome/kinome-wide RITseq coverage as depicted in Fig. Artificial chromosomes were generated Supplementary informationDataset S1Dataset S2Dataset S3Dataset S4Dataset S5