Publications by authors named "Yusuf Hannun"

347 Publications

Golgi maturation-dependent glycoenzyme recycling controls glycosphingolipid biosynthesis and cell growth via GOLPH3.

EMBO J 2021 Mar 22:e107238. Epub 2021 Mar 22.

Institute of Biochemistry and Cell Biology, National Research Council, Naples, Italy.

Glycosphingolipids are important components of the plasma membrane where they modulate the activities of membrane proteins including signalling receptors. Glycosphingolipid synthesis relies on competing reactions catalysed by Golgi-resident enzymes during the passage of substrates through the Golgi cisternae. The glycosphingolipid metabolic output is determined by the position and levels of the enzymes within the Golgi stack, but the mechanisms that coordinate the intra-Golgi localisation of the enzymes are poorly understood. Here, we show that a group of sequentially-acting enzymes operating at the branchpoint among glycosphingolipid synthetic pathways binds the Golgi-localised oncoprotein GOLPH3. GOLPH3 sorts these enzymes into vesicles for intra-Golgi retro-transport, acting as a component of the cisternal maturation mechanism. Through these effects, GOLPH3 controls the sub-Golgi localisation and the lysosomal degradation rate of specific enzymes. Increased GOLPH3 levels, as those observed in tumours, alter glycosphingolipid synthesis and plasma membrane composition thereby promoting mitogenic signalling and cell proliferation. These data have medical implications as they outline a novel oncogenic mechanism of action for GOLPH3 based on glycosphingolipid metabolism.
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http://dx.doi.org/10.15252/embj.2020107238DOI Listing
March 2021

Neutral Sphingomyelinase 2 Heightens Anti-Melanoma Immune Responses and Anti-PD-1 Therapy Efficacy.

Cancer Immunol Res 2021 Mar 16. Epub 2021 Mar 16.

INSERM UMR 1037, Cancer Research Center of Toulouse (CRCT), Toulouse, France.

Dysregulation of lipid metabolism affects the behavior of cancer cells, but how this happens is not completely understood. Neutral sphingomyelinase 2 (nSMase2), encoded by , catalyzes the breakdown of sphingomyelin to produce the anti-oncometabolite ceramide. We found that this enzyme was often downregulated in human metastatic melanoma, likely contributing to immune escape. Overexpression of nSMase2 in mouse melanoma reduced tumor growth in syngeneic wild-type but not CD8-deficient mice. In wild-type mice, nSMase2-overexpressing tumors showed accumulation of both ceramide and CD8 tumor-infiltrating lymphocytes, and this was associated with increased level of transcripts encoding IFNγ and CXCL9. Overexpressing the catalytically inactive nSMase2 failed to alter tumor growth, indicating that the deleterious effect nSMase2 has on melanoma growth depends on its enzymatic activity. , small extracellular vesicles from melanoma cells overexpressing wild-type nSMase2 augmented the expression of IL12, CXCL9, and CCL19 by bone marrow-derived dendritic cells, suggesting that melanoma nSMase2 triggers T helper 1 (Th1) polarization in the earliest stages of the immune response. Most importantly, overexpression of wild-type nSMase2 increased anti-PD-1 efficacy in murine models of melanoma and breast cancer, and this was associated with an enhanced Th1 response. Therefore, increasing expression in melanoma may serve as an original therapeutic strategy to potentiate Th1 polarization and CD8 T-cell-dependent immune responses and overcome resistance to anti-PD-1.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0342DOI Listing
March 2021

Group IIA Secreted Phospholipase A Plays a Central Role in the Pathobiology of COVID-19.

medRxiv 2021 Feb 23. Epub 2021 Feb 23.

There is an urgent need to identify cellular and molecular mechanisms responsible for severe COVID-19 disease accompanied by multiple organ failure and high mortality rates. Here, we performed untargeted/targeted lipidomics and focused biochemistry on 127 patient plasma samples, and showed high levels of circulating, enzymatically active secreted phospholipase A Group IIA (sPLA -IIA) in severe and fatal COVID-19 disease compared with uninfected patients or mild illness. Machine learning demonstrated that sPLA -IIA effectively stratifies severe from fatal COVID-19 disease. We further introduce a PLA-BUN index that combines sPLA -IIA and blood urea nitrogen (BUN) threshold levels as a critical risk factor for mitochondrial dysfunction, sustained inflammatory injury and lethal COVID-19. With the availability of clinically tested inhibitors of sPLA -IIA, our study opens the door to a precision intervention using indices discovered here to reduce COVID-19 mortality.
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http://dx.doi.org/10.1101/2021.02.22.21252237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924289PMC
February 2021

The doxorubicin-induced cell motility network is under the control of the ceramide-activated protein phosphatase 1 alpha.

FASEB J 2021 Mar;35(3):e21396

Department of Medicine, Stony Brook University, Stony Brook, NY, USA.

We have recently reported that a specific pool of ceramide, located in the plasma membrane, mediated the effects of sublethal doses of the chemotherapeutic compound doxorubicin on enhancing cancer cell migration. We identified neutral sphingomyelinase 2 (nSMase2) as the enzyme responsible to generate this bioactive pool of ceramide. In this work, we explored the role of members of the protein phosphatases 1 family (PP1), and we identified protein phosphatase 1 alpha isoform (PP1 alpha) as the specific PP1 isoform to mediate this phenotype. Using a bioinformatics approach, we build a functional interaction network based on phosphoproteomics data on plasma membrane ceramide. This led to the identification of several ceramide-PP1 alpha downstream substrates. Studies on phospho mutants of ezrin (T567) and Scrib (S1378/S1508) demonstrated that their dephosphorylation is sufficient to enhance cell migration. In summary, we identified a mechanism where reduced doses of doxorubicin result in the dysregulation of cytoskeletal proteins and enhanced cell migration. This mechanism could explain the reported effects of doxorubicin worsening cancer metastasis in animal models.
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http://dx.doi.org/10.1096/fj.202002427RDOI Listing
March 2021

Asah2 Represses the p53-Hmox1 Axis to Protect Myeloid-Derived Suppressor Cells from Ferroptosis.

J Immunol 2021 Mar 5;206(6):1395-1404. Epub 2021 Feb 5.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912;

Myeloid-derived suppressor cells (MDSCs) are immune suppressive cells that massively accumulate under pathological conditions to suppress T cell immune response. Dysregulated cell death contributes to MDSC accumulation, but the molecular mechanism underlying this cell death dysregulation is not fully understood. In this study, we report that neutral ceramidase (N-acylsphingosine amidohydrolase [ASAH2]) is highly expressed in tumor-infiltrating MDSCs in colon carcinoma and acts as an MDSC survival factor. To target ASAH2, we performed molecular docking based on human ASAH2 protein structure. Enzymatic inhibition analysis of identified hits determined NC06 as an ASAH2 inhibitor. Chemical and nuclear magnetic resonance analysis determined NC06 as 7-chloro-2-(3-chloroanilino)pyrano[3,4-e][1,3]oxazine-4,5-dione. NC06 inhibits ceramidase activity with an IC of 10.16-25.91 μM for human ASAH2 and 18.6-30.2 μM for mouse Asah2 proteins. NC06 induces MDSC death in a dose-dependent manner, and inhibition of ferroptosis decreased NC06-induced MDSC death. NC06 increases glutathione synthesis and decreases lipid reactive oxygen species to suppress ferroptosis in MDSCs. Gene expression profiling identified the p53 pathway as the Asah2 target in MDSCs. Inhibition of Asah2 increased p53 protein stability to upregulate Hmox1 expression to suppress lipid reactive oxygen species production to suppress ferroptosis in MDSCs. NC06 therapy increases MDSC death and reduces MDSC accumulation in tumor-bearing mice, resulting in increased activation of tumor-infiltrating CTLs and suppression of tumor growth in vivo. Our data indicate that ASAH2 protects MDSCs from ferroptosis through destabilizing p53 protein to suppress the p53 pathway in MDSCs in the tumor microenvironment. Targeting ASAH2 with NC06 to induce MDSC ferroptosis is potentially an effective therapy to suppress MDSC accumulation in cancer immunotherapy.
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http://dx.doi.org/10.4049/jimmunol.2000500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946776PMC
March 2021

Sphingosine kinase 1 downregulation is required for adaptation to serine deprivation.

FASEB J 2021 Feb;35(2):e21284

Department of Medicine, Stony Brook University, Stony Brook, NY, USA.

It has been well-established that cancer cells often display altered metabolic profiles, and recent work has concentrated on how cancer cells adapt to serine removal. Serine can be either taken exogenously or synthesized from glucose, and its regulation forms an important mechanism for nutrient integration. One of the several important metabolic roles for serine is in the generation of bioactive sphingolipids since it is the main substrate for serine palmitoyltransferase, the initial and rate-limiting enzyme in the synthesis of sphingolipids. Previously, serine deprivation has been connected to the action of the tumor suppressor p53, and we have previously published on a role for p53 regulating sphingosine kinase 1 (SK1), an enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P). SK1 is a key enzyme in sphingolipid synthesis that functions in pro-survival and tumor-promoting pathways and whose expression is also often elevated in cancers. Here we show that SK1 was degraded during serine starvation in a time and dose-dependent manner, which led to sphingosine accumulation. This was independent of effects on p53 but required the action of the proteasome. Furthermore, we show that overexpression of SK1, to compensate for SK1 loss, was detrimental to cell growth under conditions of serine starvation, demonstrating that the suppression of SK1 under these conditions is adaptive. Mitochondrial oxygen consumption decreased in response to SK1 degradation, and this was accompanied by an increase in intracellular reactive oxygen species (ROS). Suppression of ROS with N-acteylcysteine resulted in suppression of the metabolic adaptations and in decreased cell growth under serine deprivation. The effects of SK1 suppression on ROS were mimicked by D-erythro-sphingosine, whereas S1P was ineffective, suggesting that the effects of loss of SK1 were due to the accumulation of its substrate sphingosine. This study reveals a new mechanism for regulating SK1 levels and a link of SK1 to serine starvation as well as mitochondrial function.
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http://dx.doi.org/10.1096/fj.202001814RRDOI Listing
February 2021

Synthesis of erythro- B13 enantiomers and stereospecific action of full set of B13-isomers in MCF7 breast carcinoma cells: Cellular metabolism and effects on sphingolipids.

Bioorg Med Chem 2021 Feb 8;32:116011. Epub 2021 Jan 8.

Departments of Medicine and Biochemistry & the Stony Brook Cancer Center at Stony, Brook University, Stony Brook, NY 11794, USA. Electronic address:

B13 is an acid ceramidase (ACDase) inhibitor. The two chiral centers of this aromatic amido alcohol lead to four stereoisomers, yet we have little knowledge about its erythro- enantiomers, (1R, 2S) and (1S, 2R). In this paper, for the first time, the synthesis of two erythro- enantiomers is described, and the compounds are evaluated along with two threo- enantiomers, (1R, 2R) and (1S, 2S). The key metabolites and sphingolipid (SL) profile of the full set of B13 stereoisomers in MCF7 breast carcinoma cells are presented. The results demonstrated that the erythro- enantiomers were more effective than the threo- enantiomers on growth inhibition in MCF7 cells, although there were no statistically significant differences within the threo- and erythro- series. Measurement of intracellular levels of the compounds indicated that the erythro- seemed a little more cell permeable than the threo- enantiomers; also, the (1R, 2S) isomer with the same stereo structure as natural ceramide (Cer) could be hydrolyzed and phosphorylated in MCF7 cells. Furthermore, we also observed the formation of C16 homologs from the full set of B13 isomers within the cells, indicating the occurrence of de-acylation and re-acylation of the amino group of the aromatic alcohol. Moreover, the decrease in the Cer/Sph ratio suggests that the growth inhibition from (1R, 2S) isomer is not because of the inhibition of ceramidases. Taken together, (1R, 2S) could be developed as a substitute of natural Cer.
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http://dx.doi.org/10.1016/j.bmc.2021.116011DOI Listing
February 2021

Sphingosine-1-phosphate receptor 3 potentiates inflammatory programs in normal and leukemia stem cells to promote differentiation.

Blood Cancer Discov 2021 Jan 1;2(1):32-53. Epub 2020 Dec 1.

Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

Acute myeloid leukemia (AML) is a caricature of normal hematopoiesis, driven from leukemia stem cells (LSC) that share some hematopoietic stem cell (HSC) programs including responsiveness to inflammatory signaling. Although inflammation dysregulates mature myeloid cells and influences stemness programs and lineage determination in HSC by activating stress myelopoiesis, such roles in LSC are poorly understood. Here, we show that S1PR3, a receptor for the bioactive lipid sphingosine-1-phosphate, is a central regulator which drives myeloid differentiation and activates inflammatory programs in both HSC and LSC. S1PR3-mediated inflammatory signatures varied in a continuum from primitive to mature myeloid states across AML patient cohorts, each with distinct phenotypic and clinical properties. S1PR3 was high in LSC and blasts of mature myeloid samples with linkages to chemosensitivity, while S1PR3 activation in primitive samples promoted LSC differentiation leading to eradication. Our studies open new avenues for therapeutic target identification specific for each AML subset.
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http://dx.doi.org/10.1158/2643-3230.BCD-20-0155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116590PMC
January 2021

Ceramide synthase 2-C -ceramide axis limits the metastatic potential of ovarian cancer cells.

FASEB J 2021 Feb;35(2):e21287

Laboratory of Immunopharmacology, Setsunan University, Osaka, Japan.

Regulation of sphingolipid metabolism plays a role in cellular homeostasis, and dysregulation of these pathways is involved in cancer progression. Previously, our reports identified ceramide as an anti-metastatic lipid. In the present study, we investigated the biochemical alterations in ceramide-centered metabolism of sphingolipids that were associated with metastatic potential. We established metastasis-prone sublines of SKOV3 ovarian cancer cells using an in vivo selection method. These cells showed decreases in ceramide levels and ceramide synthase (CerS) 2 expression. Moreover, CerS2 downregulation in ovarian cancer cells promoted metastasis in vivo and potentiated cell motility and invasiveness. Moreover, CerS2 knock-in suppressed the formation of lamellipodia required for cell motility in this cell line. In order to define specific roles of ceramide species in cell motility controlled by CerS2, the effect of exogenous long- and very long-chain ceramide species on the formation of lamellipodia was evaluated. Treatment with distinct ceramides increased cellular ceramides and had inhibitory effects on the formation of lamellipodia. Interestingly, blocking the recycling pathway of ceramides by a CerS inhibitor was ineffective in the suppression of exogenous C -ceramide for the formation of lamellipodia. These results suggested that C -ceramide, a CerS2 metabolite, predominantly suppresses the formation of lamellipodia without the requirement for deacylation/reacylation. Moreover, knockdown of neutral ceramidase suppressed the formation of lamellipodia concomitant with upregulation of C -ceramide. Collectively, the CerS2-C -ceramide axis, which may be countered by neutral ceramidase, is suggested to limit cell motility and metastatic potential. These findings may provide insights that lead to further development of ceramide-based therapy and biomarkers for metastatic ovarian cancer.
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http://dx.doi.org/10.1096/fj.202001504RRDOI Listing
February 2021

Targeting sphingosine kinase 1 (SK1) enhances oncogene-induced senescence through ceramide synthase 2 (CerS2)-mediated generation of very-long-chain ceramides.

Cell Death Dis 2021 Jan 4;12(1):27. Epub 2021 Jan 4.

Department of Medicine, Stony Brook University, Stony Brook, NY, USA.

Senescence is an antiproliferative mechanism that can suppress tumor development and can be induced by oncogenes such as genes of the Ras family. Although studies have implicated bioactive sphingolipids (SL) in senescence, the specific mechanisms remain unclear. Here, using MCF10A mammary epithelial cells, we demonstrate that oncogenic K-Ras (Kirsten rat sarcoma viral oncogene homolog) is sufficient to induce cell transformation as well as cell senescence-as revealed by increases in the percentage of cells in the G1 phase of the cell cycle, p21 (p21) expression, and senescence-associated β-galactosidase activity (SA-β-gal). Furthermore, oncogenic K-Ras altered SL metabolism, with an increase of long-chain (LC) C18, C20 ceramides (Cer), and very-long-chain (VLC) C22:1, C24 Cer, and an increase of sphingosine kinase 1 (SK1) expression. Since Cer and sphingosine-1-phosphate have been shown to exert opposite effects on cellular senescence, we hypothesized that targeting SK1 could enhance oncogenic K-Ras-induced senescence. Indeed, SK1 downregulation or inhibition enhanced p21 expression and SA-β-gal in cells expressing oncogenic K-Ras and impeded cell growth. Moreover, SK1 knockdown further increased LC and VLC Cer species (C18, C20, C22:1, C24, C24:1, C26:1), especially the ones increased by oncogenic K-Ras. Fumonisin B1 (FB1), an inhibitor of ceramide synthases (CerS), reduced p21 expression induced by oncogenic K-Ras both with and without SK1 knockdown. Functionally, FB1 reversed the growth defect induced by oncogenic K-Ras, confirming the importance of Cer generation in the senescent phenotype. More specifically, downregulation of CerS2 by siRNA blocked the increase of VLC Cer (C24, C24:1, and C26:1) induced by SK1 knockdown and phenocopied the effects of FB1 on p21 expression. Taken together, these data show that targeting SK1 is a potential therapeutic strategy in cancer, enhancing oncogene-induced senescence through an increase of VLC Cer downstream of CerS2.
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http://dx.doi.org/10.1038/s41419-020-03281-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790826PMC
January 2021

Acid sphingomyelinase-dependent autophagic degradation of GPX4 is critical for the execution of ferroptosis.

Cell Death Dis 2021 Jan 7;12(1):26. Epub 2021 Jan 7.

Cell Death Signaling Laboratory, Division of Science (Biology), Experimental Research Building, New York University Abu Dhabi, P. O. Box. 129188, Abu Dhabi, UAE.

Ferroptosis is a type of regulated cell death characterized by ROS accumulation and devastating lipid peroxidation (LPO). The role of acid sphingomyelinase (ASM), a key enzyme in sphingolipid metabolism, in the induction of apoptosis has been studied; however, to date its role in ferroptosis is unclear. In this study, we report that ASM plays a hitherto unanticipated role in promoting ferroptosis. Mechanistically, Erastin (Era) treatment results in the activation of ASM and generation of ceramide, which are required for the Era-induced reactive oxygen species (ROS) generation and LPO. Inhibition of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) or removal of intracellular ROS, significantly reduced Era-induced ASM activation, suggesting that NADPH oxidase-derived ROS regulated ASM-initiated redox signaling in a positive feedback manner. Moreover, ASM-mediated activation of autophagy plays a critical role in ferroptosis inducers (FINs)-induced glutathione peroxidase 4 (GPX4) degradation and ferroptosis activation. Genetic or pharmacological inhibition of ASM diminishes Era-induced features of autophagy, GPX4 degradation, LPO, and subsequent ferroptosis. Importantly, genetic activation of ASM increases ferroptosis in cancer cells induced by various FINs. Collectively, these findings reveal that ASM plays a novel role in ferroptosis that could be exploited to improve pathological conditions that link to ferroptosis.
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http://dx.doi.org/10.1038/s41419-020-03297-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7791123PMC
January 2021

Bioactive sphingolipids: Advancements and contributions from the laboratory of Dr. Lina M. Obeid.

Cell Signal 2021 Mar 5;79:109875. Epub 2020 Dec 5.

Department of Nutritional Sciences, College of Agriculture and Life Sciences, University of Arizona, Tucson, AZ 85721, USA. Electronic address:

Sphingolipids and their synthetic enzymes have emerged as critical mediators in numerous diseases including inflammation, aging, and cancer. One enzyme in particular, sphingosine kinase (SK) and its product sphingosine-1-phosphate (S1P), has been extensively implicated in these processes. SK catalyzes the phosphorylation of sphingosine to S1P and exists as two isoforms, SK1 and SK2. In this review, we will discuss the contributions from the laboratory of Dr. Lina M. Obeid that have defined the roles for several bioactive sphingolipids in signaling and disease with an emphasis on her work defining SK1 in cellular fates and pathobiologies including proliferation, senescence, apoptosis, and inflammation.
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http://dx.doi.org/10.1016/j.cellsig.2020.109875DOI Listing
March 2021

Transcriptional Regulation of Sphingosine Kinase 1.

Cells 2020 11 8;9(11). Epub 2020 Nov 8.

Department of Pharmacology, Stony Brook University, Stony Brook, NY 11794, USA.

Once thought to be primarily structural in nature, sphingolipids have become increasingly appreciated as second messengers in a wide array of signaling pathways. Sphingosine kinase 1, or SK1, is one of two sphingosine kinases that phosphorylate sphingosine into sphingosine-1-phosphate (S1P). S1P is generally pro-inflammatory, pro-angiogenic, immunomodulatory, and pro-survival; therefore, high SK1 expression and activity have been associated with certain inflammatory diseases and cancer. It is thus important to develop an understanding of the regulation of SK1 expression and activity. In this review, we explore the current literature on SK1 transcriptional regulation, illustrating a complex system of transcription factors, cytokines, and even micro-RNAs (miRNAs) on the post transcriptional level.
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http://dx.doi.org/10.3390/cells9112437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695205PMC
November 2020

Neutral sphingomyelinase 2 regulates inflammatory responses in monocytes/macrophages induced by TNF-α.

Sci Rep 2020 10 8;10(1):16802. Epub 2020 Oct 8.

Immunology and Microbiology Department, Dasman Diabetes Institute, Al-Soor Street, P.O. Box 1180, 15462, Dasman, Kuwait, Kuwait.

Obesity is associated with elevated levels of TNF-α and proinflammatory CD11c monocytes/macrophages. TNF-α mediated dysregulation in the plasticity of monocytes/macrophages is concomitant with pathogenesis of several inflammatory diseases, including metabolic syndrome, but the underlying mechanisms are incompletely understood. Since neutral sphingomyelinase-2 (nSMase2: SMPD3) is a key enzyme for ceramide production involved in inflammation, we investigated whether nSMase2 contributed to the inflammatory changes in the monocytes/macrophages induced by TNF-α. In this study, we demonstrate that the disruption of nSMase activity in monocytes/macrophages either by chemical inhibitor GW4869 or small interfering RNA (siRNA) against SMPD3 results in defects in the TNF-α mediated expression of CD11c. Furthermore, blockage of nSMase in monocytes/macrophages inhibited the secretion of inflammatory mediators IL-1β and MCP-1. In contrast, inhibition of acid SMase (aSMase) activity did not attenuate CD11c expression or secretion of IL-1β and MCP-1. TNF-α-induced phosphorylation of JNK, p38 and NF-κB was also attenuated by the inhibition of nSMase2. Moreover, NF-kB/AP-1 activity was blocked by the inhibition of nSMase2. SMPD3 was elevated in PBMCs from obese individuals and positively corelated with TNF-α gene expression. These findings indicate that nSMase2 acts, at least in part, as a master switch in the TNF-α mediated inflammatory responses in monocytes/macrophages.
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http://dx.doi.org/10.1038/s41598-020-73912-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544688PMC
October 2020

Maternal and fetal alkaline ceramidase 2 is required for placental vascular integrity in mice.

FASEB J 2020 Nov 22;34(11):15252-15268. Epub 2020 Sep 22.

Department of Medicine, Stony Brook University, Stony Brook, NY, USA.

Sphingolipids have been implicated in mammalian placental development and function, but their regulation in the placenta remains unclear. Herein we report that alkaline ceramidase 2 (ACER2) plays a key role in sustaining the integrity of the placental vasculature by regulating the homeostasis of sphingolipids in mice. The mouse alkaline ceramidase 2 gene (Acer2) is highly expressed in the placenta between embryonic day (E) 9.5 and E12.5. Acer2 deficiency in both the mother and fetus decreases the placental levels of sphingolipids, including sphingoid bases (sphingosine and dihydrosphingosine) and sphingoid base-1-phosphates (sphingosine-1-phosphate and dihydrosphingosine-1-phosphate) and results in the in utero death of ≈50% of embryos at E12.5 whereas Acer2 deficiency in either the mother or fetus has no such effects. Acer2 deficiency causes hemorrhages from the maternal vasculature in the junctional and/or labyrinthine zones in E12.5 placentas. Moreover, hemorrhagic but not non-hemorrhagic Acer2-deficient placentas exhibit an expansion of parietal trophoblast giant cells with a concomitant decrease in the area of the fetal blood vessel network in the labyrinthine zone, suggesting that Acer2 deficiency results in embryonic lethality due to the atrophy of the fetal blood vessel network in the placenta. Taken together, these results suggest that ACER2 sustains the integrity of the placental vasculature by controlling the homeostasis of sphingolipids in mice.
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http://dx.doi.org/10.1096/fj.202001104RDOI Listing
November 2020

Targeting acid ceramidase inhibits YAP/TAZ signaling to reduce fibrosis in mice.

Sci Transl Med 2020 08;12(557)

Department of Medicine, University of California, San Francisco, San Francisco, CA 94115, USA.

Hepatic stellate cells (HSCs) drive hepatic fibrosis. Therapies that inactivate HSCs have clinical potential as antifibrotic agents. We previously identified acid ceramidase (aCDase) as an antifibrotic target. We showed that tricyclic antidepressants (TCAs) reduce hepatic fibrosis by inhibiting aCDase and increasing the bioactive sphingolipid ceramide. We now demonstrate that targeting aCDase inhibits YAP/TAZ activity by potentiating its phosphorylation-mediated proteasomal degradation via the ubiquitin ligase adaptor protein β-TrCP. In mouse models of fibrosis, pharmacologic inhibition of aCDase or genetic knockout of aCDase in HSCs reduces fibrosis, stromal stiffness, and YAP/TAZ activity. In patients with advanced fibrosis, aCDase expression in HSCs is increased. Consistently, a signature of the genes most down-regulated by ceramide identifies patients with advanced fibrosis who could benefit from aCDase targeting. The findings implicate ceramide as a critical regulator of YAP/TAZ signaling and HSC activation and highlight aCDase as a therapeutic target for the treatment of fibrosis.
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http://dx.doi.org/10.1126/scitranslmed.aay8798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7976849PMC
August 2020

Inhibition of acid ceramidase regulates MHC class II antigen presentation and suppression of autoimmune arthritis.

Cytokine 2020 Nov 29;135:155219. Epub 2020 Jul 29.

Department of Microbiology and Immunology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, United States; Darby Children's Research Institute, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, United States; Hollings Cancer Center, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, United States. Electronic address:

The bioactive sphingolipid ceramide affects immune responses although its effect on antigen (Ag) processing and delivery by HLA class II to CD4+T-cells remains unclear. Therefore, we examined the actions of a novel cell-permeable acid ceramidase (AC) inhibitor [(1R,2R) N myristoylamino-(4'-nitrophenyl)-propandiol-1,3] on antigen presentation and inflammatory cytokine production by Ag-presenting cells (APCs) such as B-cells, macrophages, and dendritic cells. We found that AC inhibition in APCs perturbed Ag-processing and presentation via HLA-DR4 (MHC class II) proteins as measured by coculture assay and T-cell production of IL-2. Mass spectral analyses showed that B13 treatment significantly raised levels of four types of ceramides in human B-cells. B13 treatment did not alter Ag internalization and class II protein expression, but significantly inhibited lysosomal cysteinyl cathepsins (B, S and L) and thiol-reductase (GILT), HLA class II Ag-processing, and generation of functional class II-peptide complexes. Ex vivo Ag presentation assays showed that inhibition of AC impaired primary and recall CD4+T-cell responses and cytokine production in response against type II collagen. Further, B13 delayed onset and reduced severity of inflamed joints and cytokine production in the collagen-induced arthritis mouse model in vivo. These findings suggest that inhibition of AC in APCs may dysregulate endolysosomal proteases and HLA class II-associated self-antigen presentation to CD4+T-cells, attenuating inflammatory cytokine production and suppressing host autoimmune responses.
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http://dx.doi.org/10.1016/j.cyto.2020.155219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502491PMC
November 2020

Identification of Small-Molecule Inhibitors of Neutral Ceramidase (nCDase) via Target-Based High-Throughput Screening.

SLAS Discov 2021 Jan 31;26(1):113-121. Epub 2020 Jul 31.

Department of Molecular Medicine, Scripps Research, The Scripps Research Molecular Screening Center, Jupiter, FL, USA.

There is interest in developing inhibitors of human neutral ceramidase (nCDase) because this enzyme plays a critical role in colon cancer. There are currently no potent or clinically effective inhibitors for nCDase reported to date, so we adapted a fluorescence-based enzyme activity method to a high-throughput screening format. We opted to use an assay whereby nCDase hydrolyzes the substrate RBM 14-16, and the addition of NaIO4 acts as an oxidant that releases umbelliferone, resulting in a fluorescent signal. As designed, test compounds that act as ceramidase inhibitors will prevent the hydrolysis of RBM 14-16, thereby decreasing fluorescence. This assay uses a 1536-well plate format with excitation in the blue spectrum of light energy, which could be a liability, so we incorporated a counterscreen that allows for rapid selection against fluorescence artifacts to minimize false-positive hits. The high-throughput screen of >650,000 small molecules found several lead series of hits. Multiple rounds of chemical optimization ensued with improved potency in terms of IC and selectivity over counterscreen assays. This study describes the first large-scale high-throughput optical screening assay for nCDase inhibitors that has resulted in leads that are now being pursued in crystal docking studies and in vitro drug metabolism and pharmacokinetics (DMPK).
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http://dx.doi.org/10.1177/2472555220945283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749003PMC
January 2021

Ceramide launches an acute anti-adhesion pro-migration cell signaling program in response to chemotherapy.

FASEB J 2020 06 20;34(6):7610-7630. Epub 2020 Apr 20.

Department of Medicine, Stony Brook University, Stony Brook, NY, USA.

Chemotherapy has been reported to upregulate sphingomylinases and increase cellular ceramide, often linked to the induction to cell death. In this work, we show that sublethal doses of doxorubicin and vorinostat still increased cellular ceramide, which was located predominantly at the plasma membrane. To interrogate possible functions of this specific pool of ceramide, we used recombinant enzymes to mimic physiological levels of ceramide at the plasma membrane upon chemotherapy treatment. Using mass spectrometry and network analysis, followed by experimental confirmation, the results revealed that this pool of ceramide acutely regulates cell adhesion and cell migration pathways with weak connections to commonly established ceramide functions (eg, cell death). Neutral sphingomyelinase 2 (nSMase2) was identified as responsible for the generation of plasma membrane ceramide upon chemotherapy treatment, and both ceramide at the plasma membrane and nSMase2 were necessary and sufficient to mediate these "side" effects of chemotherapy on cell adhesion and migration. This is the first time a specific pool of ceramide is interrogated for acute signaling functions, and the results define plasma membrane ceramide as an acute signaling effector necessary and sufficient for regulation of cell adhesion and cell migration under chemotherapeutical stress.
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http://dx.doi.org/10.1096/fj.202000205RDOI Listing
June 2020

Yeast Sphingolipid Phospholipase Gene Regulates the Spindle Checkpoint by a -Dependent Mechanism.

Mol Cell Biol 2020 05 28;40(12). Epub 2020 May 28.

The Stony Brook University Cancer Center, Stony Brook University, Stony Brook, New York, USA

Defects in the spindle assembly checkpoint (SAC) can lead to aneuploidy and cancer. Sphingolipids have important roles in many cellular functions, including cell cycle regulation and apoptosis. However, the specific mechanisms and functions of sphingolipids in cell cycle regulation have not been elucidated. Using analysis of concordance for synthetic lethality for the yeast sphingolipid phospholipase , we identified two groups of genes. The first comprises genes involved in chromosome segregation and stability (, , , , and ) as synthetically lethal with The second group, to which belongs, comprises genes involved in the spindle checkpoint (, , , and ), and they all share the same synthetic lethality with the first group. We demonstrate that spindle checkpoint genes act upstream of Isc1, and their deletion phenocopies that of Reciprocally, deletion mutants were sensitive to benomyl, indicating a SAC defect. Similar to deletion, deletion prevents spindle elongation in hydroxyurea-treated cells. Mechanistically, PP2A-Cdc55 ceramide-activated phosphatase was found to act downstream of Isc1, thus coupling the spindle checkpoint genes and Isc1 to -mediated nuclear functions.
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http://dx.doi.org/10.1128/MCB.00340-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261723PMC
May 2020

Correction: PKCα is required for Akt-mTORC1 activation in non-small cell lung carcinoma (NSCLC) with EGFR mutation.

Oncogene 2019 Nov;38(48):7366

Department of Medicine, Stony Brook University, Stony Brook, NY, 11794, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41388-019-1019-8DOI Listing
November 2019

PKCα is required for Akt-mTORC1 activation in non-small cell lung carcinoma (NSCLC) with EGFR mutation.

Oncogene 2019 11 16;38(48):7311-7328. Epub 2019 Aug 16.

Department of Medicine, Stony Brook University, Stony Brook, NY, 11794, USA.

Mutational activation of the epidermal growth factor receptor (EGFR) is a major player in the pathogenesis of non-small cell lung cancer (NSCLC). NSCLC patients with constitutively active EGFR mutations eventually develop drug resistance against EGFR tyrosine-kinase inhibitors; therefore, better understandings of key components of mutant EGFR (mtEGFR) signaling are required. Here, we initially observed aberrantly high expression of protein kinase Cα (PKCα) in lung adenocarcinomas, especially those with EGFR mutations, and proceeded to examine the role of PKCα in the regulation of the signaling pathways downstream of mtEGFR. The results showed that NSCLC cell lines with constitutively active EGFR mutations tend to have very or moderately high PKCα levels. Furthermore, PKCα was constitutively activated in HCC827 and H4006 cells which have an EGFR deletion mutation in exon 19. Interestingly, mtEGFR was not required for the induction of PKCα at protein and message levels suggesting that the increased levels of PKCα are due to independent selection. On the other hand, mtEGFR activity was required for robust activation of PKCα. Loss of functions studies revealed that the NSCLC cells rely heavily on PKCα for the activation of the mTORC1 signaling pathway. Unexpectedly, the results demonstrated that PKCα was required for activation of Akt upstream of mTOR but only in cells with the mtEGFR and with the increased expression of PKCα. Functionally, inhibition of PKCα in HCC827 led to caspase-3-dependent apoptosis and a significant decrease in cell survival in response to cellular stress induced by serum starvation. In summary, the results identified important roles of PKCα in regulating mTORC1 activity in lung cancer cells, whereby a primary switching occurs from PKCα-independent to PKCα-dependent signaling in the presence of EGFR mutations. The results present PKCα as a potential synergistic target of personalized treatment for NSCLC with constitutively active mutant forms of EGFR and constitutively active PKCα.
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http://dx.doi.org/10.1038/s41388-019-0950-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883150PMC
November 2019

Probing compartment-specific sphingolipids with targeted bacterial sphingomyelinases and ceramidases.

J Lipid Res 2019 11 26;60(11):1841-1850. Epub 2019 Jun 26.

Department of Medicine and Stony Brook Cancer Center, Stony Brook University, Stony Brook, NY

Sphingolipids contribute to the regulation of cell and tissue homeostasis, and disorders of sphingolipid metabolism lead to diseases such as inflammation, stroke, diabetes, and cancer. Sphingolipid metabolic pathways involve an array of enzymes that reside in specific subcellular organelles, resulting in the formation of many diverse sphingolipids with distinct molecular species based on the diversity of the ceramide (Cer) structure. In order to probe compartment-specific metabolism of sphingolipids in this study, we analyzed the Cer and SM species preferentially produced in the inner plasma membrane (PM), Golgi apparatus, ER, mitochondria, nucleus, and cytoplasm by using compartmentally targeted bacterial SMases and ceramidases. The results showed that the length of the acyl chain of Cer becomes longer according to the progress of Cer from synthesis in the ER to the Golgi apparatus, then to the PM. These findings suggest that each organelle shows different properties of SM-derived Cers consistent with its emerging distinct functions in vitro and in vivo.
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http://dx.doi.org/10.1194/jlr.M094722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824496PMC
November 2019

Bioactive sphingolipid profile in a xenograft mouse model of head and neck squamous cell carcinoma.

PLoS One 2019 19;14(4):e0215770. Epub 2019 Apr 19.

Departments of Medicine and Biochemistry & the Stony Brook Cancer Center at Stony Brook University, Stony Brook, New York, United States of America.

The purpose of this study was to determine the profile of bioactive sphingolipids in xenograft mouse tissues of head and neck squamous cell carcinoma. We utilized UHPLC-MS/MS to determine the profile of full set of ceramides, sphingosine, and sphingosine 1-phosphate in this xenograft mouse model. The tissues isolated and investigated were from brain, lung, heart, liver, spleen, kidney, bladder, tumors and blood. With the exception of equal volume of blood plasma (100ul), all tissues were studied with the same amount of protein (800ug). Results demonstrated that brain contained the highest level of ceramide and kidney had the highest level of sphingosine, whereas sphingosine 1-phosphate and dihydrosphingosine 1-phosphate were heavily presented in the blood. Brain also comprised the highest level of phospholipids. As for the species, several ceramides, usually present in very low amounts in cultured tumor cells, showed relatively high levels in certain tissues. This study highlights levels of bioactive sphingolipids profiles in xenograft mouse model of head and neck squamous cell carcinoma, and provides resources to investigate potential therapeutic targets and biomarkers that target bioactive sphingolipids metabolism pathways.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0215770PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6474618PMC
January 2020

Approaches for probing and evaluating mammalian sphingolipid metabolism.

Anal Biochem 2019 06 24;575:70-86. Epub 2019 Mar 24.

Department of Medicine, Stony Brook University, Stony Brook, NY, USA; The Stony Brook Cancer Center, Stony Brook University, Stony Brook, NY, USA; Departments of Biochemistry, Pathology and Pharmacology, Stony Brook University, Stony Brook, NY, USA. Electronic address:

Sphingolipid metabolism plays a critical role in regulating processes that control cellular fate. This dynamic pathway can generate and degrade the central players: ceramide, sphingosine and sphingosine-1-phosphate in almost any membrane in the cell, adding an unexpected level of complexity in deciphering signaling events. While in vitro assays have been developed for most enzymes in SL metabolism, these assays are setup for optimal activity conditions and can fail to take into account regulatory components such as compartmentalization, substrate limitations, and binding partners that can affect cellular enzymatic activity. Therefore, many in-cell assays have been developed to derive results that are authentic to the cellular situation which may give context to alteration in SL mass. This review will discuss approaches for utilizing probes for mammalian in-cell assays to interrogate most enzymatic steps central to SL metabolism. The use of inhibitors in conjunction with these probes can verify the specificity of cellular assays as well as provide valuable insight into flux in the SL network. The use of inhibitors specific to each of the central sphingolipid enzymes are also discussed to assist researchers in further interrogation of these pathways.
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http://dx.doi.org/10.1016/j.ab.2019.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498843PMC
June 2019

The juxtamembrane linker in neutral sphingomyelinase-2 functions as an intramolecular allosteric switch that activates the enzyme.

J Biol Chem 2019 05 19;294(18):7488-7502. Epub 2019 Mar 19.

From the Departments of Biochemistry and Cell Biology,

Neutral sphingomyelinase 2 (nSMase2) produces the bioactive lipid ceramide and has important roles in neurodegeneration, cancer, and exosome formation. Although nSMase2 has low basal activity, it is fully activated by phosphatidylserine (PS). Previous work showed that interdomain interactions within nSMase2 are needed for PS activation. Here, we use multiple approaches, including small angle X-ray scattering, hydrogen-deuterium exchange-MS, circular dichroism and thermal shift assays, and membrane yeast two-hybrid assays, to define the mechanism mediating this interdomain interactions within nSMase2. In contrast to what we previously assumed, we demonstrate that PS binding at the N-terminal and juxtamembrane regions of nSMase2 rather acts as a conformational switch leading to interdomain interactions that are critical to enzyme activation. Our work assigns a unique function for a class of linkers of lipid-activated, membrane-associated proteins. It indicates that the linker actively participates in the activation mechanism via intramolecular interactions, unlike the canonical linkers that typically aid protein dimerization or localization.
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http://dx.doi.org/10.1074/jbc.RA118.007288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509483PMC
May 2019

Quantifying 1-deoxydihydroceramides and 1-deoxyceramides in mouse nervous system tissue.

Prostaglandins Other Lipid Mediat 2019 04 19;141:40-48. Epub 2019 Feb 19.

Health Science Center, L-4, 179, Stony Brook University Medical Center, Stony Brook, NY, 11794-8430, United States. Electronic address:

Accumulation of deoxysphingolipids (deoxySLs) has been implicated in many neural diseases, although mechanisms remain unclear. A major obstacle limiting understanding of deoxySLs has been the lack of a method easily defining measurement of deoxydihydroceramide (deoxydhCer) and deoxyceramide (deoxyCer) in neural tissues. Furthermore, it is poorly understood if deoxySLs accumulate in the nervous system with aging. To facilitate investigation of deoxydhCer and deoxyCer in nervous system tissue, we developed a method to evaluate levels of these lipids in mouse brain, spinal cord, and sciatic nerve. Many deoxydhCers and brain C24-deoxyCer were present at 1, 3, and 6 months of age. Furthermore, while ceramide levels decreased with age, deoxydhCers increased in sciatic nerve and spinal cord, suggesting they may accumulate in peripheral nerves. C22-deoxydhCer was the highest deoxydhCer species in all tissues, suggesting it may be important physiologically. The development of this method will facilitate straightforward profiling of deoxydhCers and deoxyCers and the study of their metabolism and function. These results also reveal that deoxydhCers accumulate in peripheral nerves with normal aging.
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http://dx.doi.org/10.1016/j.prostaglandins.2019.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467697PMC
April 2019

Emergence of membrane sphingolipids as a potential therapeutic target.

Biochimie 2019 Mar 28;158:257-264. Epub 2019 Jan 28.

State Key Laboratory of Biocontrol, Guangdong Key Laboratory of Plant Resources, School of Life Science, Sun Yat-sen University, Guangzhou, 510275, China. Electronic address:

Background: Though sphingolipids are ubiquitously present in eukaryotic cells, but until the last decade, they were merely considered as a structural component of the plasma membrane with limited function. However, over the last decade, numerous functions have been ascribed to sphingolipids after the seminal discoveries on the bioactivities of several sphingolipids.

Scope Of Review: Sphingolipids are now well-recognized signals for fundamental cellular processes. Here we discussed about the advent of several sphingolipids components as potential therapeutic target for both human and plants.

Major Conclusions: Sphingolipid contents and/or sphingolipid-metabolizing enzyme expression/activity often get impaired during pathophysiological conditions, and hence manipulation of this signaling pathway may be beneficial in disease diagnosis, and the plasma concentrations can serve as an important prognostic and diagnostic marker for the disease.

General Significance: Sphingolipids are emerging as a goldmine for new therapeutic drug targets with promising new applications (cosmeceutical and nutraceutical), thereby opening new avenues for pharmaceuticals and nutraceutical industries.
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http://dx.doi.org/10.1016/j.biochi.2019.01.018DOI Listing
March 2019

RPGRIP1L is required for stabilizing epidermal keratinocyte adhesion through regulating desmoglein endocytosis.

PLoS Genet 2019 01 28;15(1):e1007914. Epub 2019 Jan 28.

Department of Pathology, Stony Brook University, Stony Brook, NY, United States of America.

Cilia-related proteins are believed to be involved in a broad range of cellular processes. Retinitis pigmentosa GTPase regulator interacting protein 1-like (RPGRIP1L) is a ciliary protein required for ciliogenesis in many cell types, including epidermal keratinocytes. Here we report that RPGRIP1L is also involved in the maintenance of desmosomal junctions between keratinocytes. Genetically disrupting the Rpgrip1l gene in mice caused intraepidermal blistering, primarily between basal and suprabasal keratinocytes. This blistering phenotype was associated with aberrant expression patterns of desmosomal proteins, impaired desmosome ultrastructure, and compromised cell-cell adhesion in vivo and in vitro. We found that disrupting the RPGRIP1L gene in HaCaT cells, which do not form primary cilia, resulted in mislocalization of desmosomal proteins to the cytoplasm, suggesting a cilia-independent function of RPGRIP1L. Mechanistically, we found that RPGRIP1L regulates the endocytosis of desmogleins such that RPGRIP1L-knockdown not only induced spontaneous desmoglein endocytosis, as determined by AK23 labeling and biotinylation assays, but also exacerbated EGTA- or pemphigus vulgaris IgG-induced desmoglein endocytosis. Accordingly, inhibiting endocytosis with dynasore or sucrose rescued these desmosomal phenotypes. Biotinylation assays on cell surface proteins not only reinforced the role of RPGRIP1L in desmoglein endocytosis, but also suggested that RPGRIP1L may be more broadly involved in endocytosis. Thus, data obtained from this study advanced our understanding of the biological functions of RPGRIP1L by identifying its role in the cellular endocytic pathway.
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http://dx.doi.org/10.1371/journal.pgen.1007914DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366717PMC
January 2019