scholarly journals The human UDP-galactose 4’-epimerase (GALE) is required for cell surface glycome structure and function

2019 ◽  
Author(s):  
Alex Broussard ◽  
Alyssa Florwick ◽  
Chelsea Desbiens ◽  
Nicole Nischan ◽  
Corrina Robertson ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fas Ligand ◽  
Death Receptor ◽  
Cell Physiology ◽  
Uridine Diphosphate ◽  
Death Receptor Signaling ◽  
Nucleotide Sugars ◽  
Induced Apoptosis ◽  
And Function

ABSTRACTGlycan biosynthesis relies on nucleotide-sugars (NS), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo, signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease, but how mammalian cells regulate NS levels and pathway flux remains largely uncharacterized. To address this knowledge gap, we examined uridine diphosphate (UDP)-galactose 4’-epimerase (GALE), which interconverts two pairs of essential NSs. GALE deletion in human cells triggered major imbalances in its substrate NSs and consequent dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the Fas death receptor. NS dysregulation also directly impacted cell signaling, as GALE−/− cells exhibit Fas hypoglycosylation and hypersensitivity to Fas ligand-induced apoptosis. Our results reveal a new role for GALE-mediated NS regulation in supporting death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome.

scholarly journals Human UDP-galactose 4′-epimerase (GALE) is required for cell-surface glycome structure and function

2019 ◽  
Vol 295 (5) ◽  
pp. 1225-1239
Author(s):  
Alex Broussard ◽  
Alyssa Florwick ◽  
Chelsea Desbiens ◽  
Nicole Nischan ◽  
Corrina Robertson ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Antigen ◽  
Mammalian Cells ◽  
Death Receptor ◽  
Cell Physiology ◽  
Total Sialic Acid ◽  
Death Receptor Signaling ◽  
Induced Apoptosis ◽  
And Function

Glycan biosynthesis relies on nucleotide sugars (NSs), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo, signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease. However, how mammalian cells regulate NS levels and pathway flux remains largely uncharacterized. To address this knowledge gap, here we examined UDP-galactose 4′-epimerase (GALE), which interconverts two pairs of essential NSs. Using immunoblotting, flow cytometry, and LC-MS–based glycolipid and glycan profiling, we found that CRISPR/Cas9-mediated GALE deletion in human cells triggers major imbalances in NSs and dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the cell-surface death receptor FS-7-associated surface antigen. In particular, we observed substantial decreases in total sialic acid, galactose, and GalNAc levels in glycans. These changes also directly impacted cell signaling, as GALE−/− cells exhibited FS-7-associated surface antigen ligand-induced apoptosis. Our results reveal a role of GALE-mediated NS regulation in death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome.

scholarly journals Hexokinases inhibit death receptor–dependent apoptosis on the mitochondria

2021 ◽  
Vol 118 (33) ◽  
pp. e2021175118
Author(s):  
Joachim Lauterwasser ◽  
Franziska Fimm-Todt ◽  
Aline Oelgeklaus ◽  
Annabell Schreiner ◽  
Kathrin Funk ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Specific Resistance ◽  
Resistance Mechanism ◽  
Death Receptor ◽  
Protective Mechanism ◽  
Apoptosis Pathway ◽  
Mitochondrial Apoptosis Pathway ◽  
Death Receptor Signaling ◽  
Glucose Phosphorylation ◽  
Induced Apoptosis

Death receptor–mediated apoptosis requires the mitochondrial apoptosis pathway in many mammalian cells. In response to death receptor signaling, the truncated BH3-only protein BID can activate the proapoptotic BCL-2 proteins BAX and BAK and trigger the permeabilization of the mitochondria. BAX and BAK are inhibited by prosurvival BCL-2 proteins through retrotranslocation from the mitochondria into the cytosol, but a specific resistance mechanism to truncated BID-dependent apoptosis is unknown. Here, we report that hexokinase 1 and hexokinase 2 inhibit the apoptosis activator truncated BID as well as the effectors BAX and BAK by retrotranslocation from the mitochondria into the cytosol. BCL-2 protein shuttling and protection from TRAIL- and FasL-induced cell death requires mitochondrial hexokinase localization and interactions with the BH3 motifs of BCL-2 proteins but not glucose phosphorylation. Together, our work establishes hexokinase-dependent retrotranslocation of truncated BID as a selective protective mechanism against death receptor–induced apoptosis on the mitochondria.

scholarly journals Glutathione peroxidase-1 regulates ASK1-dependent apoptosis via interaction with TRAF2 in RIPK3-negative cancer cells

2021 ◽  
Author(s):  
Sunmi Lee ◽  
Eun-Kyung Lee ◽  
Dong Hoon Kang ◽  
Jiyoung Lee ◽  
Soo Hyun Hong ◽  
...  
Keyword(s):  
Glutathione Peroxidase ◽  
Cancer Cells ◽  
Mammalian Cells ◽  
Apoptosis Pathway ◽  
Glutathione Peroxidase 1 ◽  
Peroxidase Enzyme ◽  
Thioredoxin 1 ◽  
Sequential Activation ◽  
Induced Apoptosis

AbstractGlutathione peroxidase (GPx) is a selenocysteine-containing peroxidase enzyme that defends mammalian cells against oxidative stress, but the role of GPx signaling is poorly characterized. Here, we show that GPx type 1 (GPx1) plays a key regulatory role in the apoptosis signaling pathway. The absence of GPx1 augmented TNF-α-induced apoptosis in various RIPK3-negative cancer cells by markedly elevating the level of cytosolic H2O2, which is derived from mitochondria. At the molecular level, the absence of GPx1 led to the strengthened sequential activation of sustained JNK and caspase-8 expression. Two signaling mechanisms are involved in the GPx1-dependent regulation of the apoptosis pathway: (1) GPx1 regulates the level of cytosolic H2O2 that oxidizes the redox protein thioredoxin 1, blocking ASK1 activation, and (2) GPx1 interacts with TRAF2 and interferes with the formation of the active ASK1 complex. Inducible knockdown of GPx1 expression impaired the tumorigenic growth of MDA-MB-231 cells (>70% reduction, P = 0.0034) implanted in mice by promoting apoptosis in vivo. Overall, this study reveals the apoptosis-related signaling function of a GPx family enzyme highly conserved in aerobic organisms.

Differential protective effects of Bcl-xL and Bcl-2 on apoptotic liver injury in transgenic mice

1999 ◽  
Vol 277 (3) ◽  
pp. G702-G708 ◽  
Author(s):  
Alix de la Coste ◽  
Monique Fabre ◽  
Nathalie McDonell ◽  
Arlette Porteu ◽  
Helène Gilgenkrantz ◽  
...  
Keyword(s):  
Liver Injury ◽  
Transgenic Mice ◽  
Fas Ligand ◽  
Dependent Manner ◽  
Protective Effects ◽  
Tnf Α ◽  
Apoptotic Pathways ◽  
Induced Apoptosis ◽  
Factor Α

Fas ligand (CD95L) and tumor necrosis factor-α (TNF-α) are pivotal inducers of hepatocyte apoptosis. Uncontrolled activation of these two systems is involved in several forms of liver injury. Although the broad antiapoptotic action of Bcl-2 and Bcl-xL has been clearly established in various apoptotic pathways, their ability to inhibit the Fas/CD95- and TNF-α-mediated apoptotic signal has remained controversial. We have demonstrated that the expression of BCL-2 in hepatocytes protects them against Fas-induced fulminant hepatitis in transgenic mice. The present study shows that transgenic mice overexpressing[Formula: see text]in hepatocytes are also protected from Fas-induced apoptosis in a dose-dependent manner. Bcl-xL and Bcl-2 were protective without any change in the level of endogenous[Formula: see text]or Bax and inhibited hepatic caspase-3-like activity. In vivo injection of TNF-α caused massive apoptosis and death only when transcription was inhibited. Under these conditions,[Formula: see text]mice were partially protected from liver injury and death but PK-BCL-2 mice were not. A similar differential protective effect of Bcl-xL and Bcl-2 transgenes was observed when Fas/CD95 was activated and transcription blocked. These results suggest that apoptosis triggered by activation of both Fas/CD95 and TNF-α receptors is to some extent counteracted by the transcription-dependent protective effects, which are essential for the antiapoptotic activity of Bcl-2 but not of Bcl-xL. Therefore, Bcl-xL and Bcl-2 appear to have different antiapoptotic effects in the liver whose characterization could facilitate their use to prevent the uncontrolled apoptosis of hepatocytes.

scholarly journals A Novel High-Throughput Technique for Identifying Monoclonal Antibodies capable of Death Receptor Induced Apoptosis

2009 ◽  
Vol 2 ◽  
pp. JCD.S3660
Author(s):  
Hang Fai Kwok ◽  
Julie A. Gormley ◽  
Christopher J. Scott ◽  
James A. Johnston ◽  
Shane A. Olwill
Keyword(s):  
Cell Death ◽  
High Throughput ◽  
High Throughput Screening ◽  
False Negative ◽  
Death Receptor ◽  
Annexin V ◽  
Fas Receptor ◽  
Induced Apoptosis

The study of death receptor family induced apoptosis has gained momentum in recent years with the knowledge that therapeutic antibodies targeting DR4 and DR5 (death receptor's 4 and 5) have proved efficacious in multiple clinical trials. The therapeutic rationale is based on targeting and amplifying a tumour tissues normal cell death programme (apoptosis). While advances in the targeting of DR4 and DR5 have been successful the search for an agonistic antibody to another family member, the Fas receptor, has proven more elusive. This is partly due to the differing in vitro and in vivo characteristics of individual antibodies. In order to induce Fas targeted cell death an antibody must be capable of binding to and trimerising the receptor. It has been shown that antibodies capable of performing this function in vivo, with the assistance of tumour associated cells, do not always induce apoptosis in vitro. As a result the use of current methodologies to detect functional antibodies in vitro may have dismissed potential therapeutic candidates ('false negative'). Here we report a novel high throughput screening technique which artificially cross-links antibodies bound to the Fas receptor. By combining this process with Annexin-V and Prodidium Iodide (PI) staining we can select for antibodies which have the potential to induce apoptosis in vivo.

Surprising roles for phospholipid binding proteins revealed by high throughput geneticsThis paper is one of a selection of papers published in this special issue entitled “Second International Symposium on Recent Advances in Basic, Clinical, and Social Medicine” and has undergone the Journal's usual peer review process.

2010 ◽  
Vol 88 (4) ◽  
pp. 565-574 ◽  
Author(s):  
Marissa A. LeBlanc ◽  
Christopher R. McMaster
Keyword(s):  
High Throughput ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Essential Gene ◽  
Peer Review Process ◽  
Vesicular Transport ◽  
Lipid Binding ◽  
And Function ◽  
Lipid Binding Proteins

Saccharomyces cerevisiae remains an ideal organism for studying the cell biological roles of lipids in vivo, as yeast has phospholipid metabolic pathways similar to mammalian cells, is easy and economical to manipulate, and is genetically tractable. The availability of isogenic strains containing specific genetic inactivation of each non-essential gene allowed for the development of a high-throughput method, called synthetic genetic analysis (SGA), to identify and describe precise pathways or functions associated with specific genes. This review describes the use of SGA to aid in elucidating the function of two lipid-binding proteins that regulate vesicular transport, Sec14 and Kes1. Sec14 was first identified as a phosphatidylcholine (PC) – phosphatidylinositol (PI) transfer protein required for viability, with reduced Sec14 function resulting in diminished vesicular transport out of the trans-Golgi. Although Sec14 is required for cell viability, inactivating the KES1 gene that encodes for a member of the oxysterol binding protein family in cells lacking Sec14 function results in restoration of vesicular transport and cell growth. SGA analysis identified a role for Kes1 and Sec14 in regulating the level and function of Golgi PI-4-phosphate (PI-4-P). SGA also determined that Sec14 not only regulates vesicular transport out of the trans-Golgi, but also transport from endosomes to the trans-Golgi. Comparing SGA screens in databases, coupled with genetic and cell biological analyses, further determined that the PI-4-P pool affected by Kes1 is generated by the PI 4-kinase Pik1. An important biological role for Sec14 and Kes1 revealed by SGA is coordinate regulation of the Pik1-generated Golgi PI-4-P pool that in turn is essential for vesicular transport into and out of the trans-Golgi.

Chromatin structure and function: the heretical path to an RNA transcription factor

1996 ◽  
Vol 74 (5) ◽  
pp. 623-632 ◽  
Author(s):  
Margarida O. Krause
Keyword(s):  
Chromatin Structure ◽  
Mammalian Cells ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Mobility Shift ◽  
Cell Extracts ◽  
Myc Gene ◽  
And Function

This review represents a synthesis of the work of the author and her collaborators through 40 years of research aimed at an understanding of chromatin composition and functional arrangement. It describes the progressive experimental stages, starting with autoradiography and protein analysis and continuing on to a more functional approach testing the template properties of intact nuclei, as well as nuclei depleted of, or reconstituted with, defined fractions extracted from the chromatin of other cell lines or tissues. As new questions were raised at each phase of these studies, the investigation was shifted from chromosomal proteins to the role of a small RNA that coextracted with one protein fraction and whose properties suggested a transcription-activating function. The active RNA was identified as a class in RNA, designated as 7 SK. Its properties suggested a role in the activation of two oncogenes, the SV 40 T-antigen and the mammalian c-myc gene. A detailed analysis of the c-myc gene expression during transformation induction in temperature-sensitive mammalian cells finally culminated in in vivo evidence for a role of 7 SK in c-myc deregulation, using cells transfected with antisense oligonucleotides to block 7 SK activity. This was followed by an investigation of promoter targeting by 7 SK RNP using electrophoretic mobility shift assays with whole or 7 SK-depleted cell extracts. Taken together, these studies indicate that 7 SK RNP participates in transformation-dependent deregulation of the c-myc gene by activation of two c-myc minor promoters. The implications of these findings are discussed.Key words: chromatin structure, histones, nonhistones, 7 SK RNA, the c-myc gene, transcription regulation, SV 40, transformation.

A Phase I Study of Immune Gene Therapy for Patients with CLL Using a Membrane-Stable, Humanized CD154.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2040-2040 ◽  
Author(s):  
William G. Wierda ◽  
J. Castro ◽  
R. Aguillon ◽  
A. Jalayer ◽  
J. McMannis ◽  
...  
Keyword(s):  
T Cell ◽  
Phase I ◽  
Death Receptor ◽  
Leukemia Cells ◽  
Cell Counts ◽  
Post Infusion ◽  
Bystander Cells ◽  
Induced Apoptosis ◽  
Dose Levels

Abstract Chronic lymphocytic leukemia (CLL) is an ideal disease for therapeutic vaccine strategies. While the leukemia cells are usually stealth-like, avoiding T cell recognition, they can be manipulated through ligation of CD40 on their surface to become very effective antigen presenting cells (APCs). Ligation of CD40 leads to expression of CD80, CD86 and upregulation of CD54. Other biochemical changes occur upon ligation of CD40, including upregulation of CD95, DR5, and expression of Bid, predisposing the leukemia cells to death-receptor-induced apoptosis. CLL cells can be made to express CD154 (CD40-ligand) using a replication-defective adenovirus. A phase I clinical trial with autologous CLL cells transduced to express murine CD154 previously demonstrated tolerability and clinical activity with this strategy (Blood96:2917, 2000). More recently, a recombinant CD154 (ISF35) was produced, based on the human CD154 backbone, incorporating murine sequences needed for expression on CLL cells and with the proteinase cleavage site removed. We evaluated this new transgene in a phase I clinical trial, expecting to have similar tolerability to the murine CD154. Transduction of CLL cells results in expression of ISF35, ligation of CD40 on transduced and bystander cells, and the resultant downstream changes needed for antigen presentation and sensitivity to death receptor-induced apoptosis. We conducted a phase I study of a single infusion of autologous CLL cells transduced to express ISF35. Three dose levels were evaluated with 3 patients (pts) each: 1×108, 3×108, & 1×109 transduced cells. Infusions were well tolerated, no acute infusion-related toxicities were observed. ISF35-related toxicities consisted of grade 1–2 flu-like symptoms that occurred several hrs after the infusion and consisted of fever, arthralgia, myalgia, nausea, vomiting, and fatigue lasting 2–4 days and resolving in all cases. There were no dose-limiting toxicities at any dose level. Biologic responses were seen at all doses, there was no dose-response relationship. There were consistent decreases in absolute lymphocyte counts at all dose levels, indicating a therapeutic effect. This was not dose-related, and ALC returned to pre-treatment level after 1–2 months post-infusion. There was consistent induction of CD95 and DR5 expression on bystander cells in vivo by 3 days following infusion, which lasted 2–3 weeks. Furthermore, consistent induction of Bid expression in bystander cells was seen by wk 1, also lasting 2–3 wks. Finally, consistent increases in absolute T cell counts (both CD4+ & CD8+) were seen, peaking 1–4 wks post infusion. These results demonstrate that ISF34-transduced autologous leukemia cells can be given safely at up to 1×109 transduced cells, without dose-limiting toxicities, and resulting in phenotypic and biochemical changes in bystander leukemia cells in vivo that render the cells able to present antigen and priming them for death-receptor-induced apoptosis. Furthermore, clinical responses were seen with reduction in leukemia counts and increases in absolute T cell counts. We expect that multiple, sequential doses will be needed for maximal therapeutic effect with this strategy. Given these results, we have developed a phase II trial of repeated doses of autologous ISF35-transduced leukemia cells for patients with CLL.

Acquired Resistance to Tumor Necrosis Factor-Related Apoptosis- Inducing Ligand (TRAIL): Histone Deacetylase Inhibitors Resensitize TRAIL-Resistant Jurkat Acute Lymphocytic Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5032-5032
Author(s):  
Pavel Klener ◽  
Jan Molinsky ◽  
Tereza Simonova ◽  
Emanuel Necas ◽  
Ladislav Andera ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Fas Ligand ◽  
Death Receptor ◽  
Lymphocytic Leukemia ◽  
Jurkat Cells ◽  
Tnf Alpha ◽  
Apoptotic Pathway ◽  
Induced Apoptosis ◽  
Disc Formation

Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a death-ligand from the TNF family. TRAIL induces programmed cell death by the cell-extrinsic p53-independent apoptotic pathway. A potential of TRAIL as cancer-specific therapeutic agent has been proposed and is preclinically and clinically tested. Development of TRAIL-resistant clones in the TRAIL-sensitive tumor cells may be a serious complication of TRAIL based cancer therapy. Jurkat acute lymphocytic leukemia cells are sensitive to TRAIL-induced apoptosis, as well as other apoptosis inducing ligands from TNF family, Fas and TNF-alpha. Jurkat cells express only one of the four receptors for TRAIL, death receptor 5 (DR5). Prolonged exposure of TRAIL-sensitive Jurkat cells to recombinant soluble TRAIL (1000 ng/mL) resulted in the establishment of three TRAIL-resistant (TR) Jurkat cell subclones, Jurkat TR1, TR2, and TR3. The Jurkat TR subclones were also resistant to TNF-alpha and Fas ligand, suggesting disruption of the extrinsic apoptotic pathway. TRAIL-resistant subclone TR1, but not TR2 and TR3, demonstrated decreased susceptibility to undergo apoptosis in response to histone-deacetylase inhibitors, valproic acid (VA), sodium butyrate (SB) and suberoylanilide hydroxamic acid (SAHA) and was resistant to fludarabine. Flow cytometry analysis showed Jurkat TR subclones had unchanged expression of cell surface death receptor DR5, Fas, and receptors for TNF-alpha, TNF-R1 and TNF-R2, compared to TRAIL-sensitive Jurkat cells. Analysis of death-inducing signaling complex (DISC) formation by immunoprecipitation (anti-TRAIL, anti-DR5) and subsequent western blotting (anti-caspase 8, anti-FADD) clearly demonstrated that the DISC formation in response to TRAIL binding to DR5 was significantly decreased in subclones TR2 and TR3, but remained unchanged in subclone TR1 compared to TRAIL-sensitive Jurkat cells. To gain further insight into potential molecular aletarations associated with acquired TRAIL resistance of Jurkat subclones, we measured gene expression of several key apoptotic regulators, including receptors for TRAIL, cFLIP, BCL2 family, IAP family, HSP family members in TRAIL-resistant and TRAIL-sensitive Jurkat cells and did not detect any significant (>2-fold) change. These results suggest acquired TRAIL resistance of Jurkat cells might be mediated by changes on the protein rather than mRNA level. We analyzed whether the TRAIL-resistant Jurkat cells could be resensitized to TRAIL-induced apoptosis by pretreatment with diverse inhibitors of important prosurvival pathways, including inhibitors of proteosynthesis (cycloheximid), inhibitors of transcription (actinomycin D), NFkB inhibitors (bortezomib, SN-50), PI3K-Akt-mTOR inhibitors (rapamycine, LY294002, Hsp90 inhibitor (17-AAG), cyclin-dependent kinase inhibitors (roscovitine), casein kinase II inhibitors (DRB), or histone deacetylase inhibitors (HDACi: SAHA, VA, SB). Pretreatment with HDAC inhibitors for 12 hour was able to resensitize all three TRAIL-resistant Jurkat subclones to TRAIL-induced apoptosis. The percentage of apoptotic cells of HDACi-pretreated subclones was 70–95% 24 h after the exposure to TRAIL compared to 5–15% apoptosis for HDACi-untreated TRAIL-exposed controls, and to 10–15% apoptosis for HDACi-treated TRAIL unexposed controls. We established TRAIL-resistant subclones from the original TRAIL-sensitive Jurkat cells. Acquired resistance to TRAIL was not mediated by downregulation of TRAIL death receptor DR5 and was associated with (cross)resistance to TNFa and Fas ligand, suggesting disruption of cell-extrinsic apoptotic pathway. We assume diverse molecular mechanisms were involved in the development of TRAIL-resistant subclones upon exposure to TRAIL, as exemplified by disrupted formation of DISC in case of subclones TR2 and TR3 and normal DISC formation and fludarabine resistance in subclone TR1, suggesting deregulated apoptotic pathway downstream of DISC. Finally, we observed that HDACi resensitized the TRAIL-resistant subclones to TRAIL. The results provide substantiation for combinatorial approaches in the potential TRAIL-based therapies of hematological malignancies.

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