Identification of a novel amidase motif in neutral ceramidase

Galadari, Sehamuddin; Wu, Bill X.; Mao, Cungui; Roddy, Patrick; Bawab, Samer El; Hannun, Yusuf A.

doi:10.1042/bj20050682

Cited by 44 publications

(50 citation statements)

References 40 publications

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“…However, there were no significant differences in the activity of acid, neutral, and alkaline ceramidases (43-47) between K562 and K562-IMA-1 cells, as measured in vitro using total cell lysates (data not shown), as described previously (43)(44)(45)(46)(47). Nevertheless, although these data may suggest that alterations of ceramidase (CDase) enzyme activity might not be involved in the development of resistance to imatinib, inhibition of a specific CDase, which might preferentially utilize LASS1-generated C 18 -ceramide as a substrate, can still provide a novel tool to improve the efficacy of imatinib and reverse resistance to this drug.…”

Section: Discussionsupporting

confidence: 58%

“…It should also be noted that increased activity/expression of one or more ceramidases, required for the hydrolysis of ceramide to generate sphingosine (43)(44)(45)(46)(47), which is then converted to S1P by SK1, might also be involved in the development of resistance to imatinib. However, there were no significant differences in the activity of acid, neutral, and alkaline ceramidases (43-47) between K562 and K562-IMA-1 cells, as measured in vitro using total cell lysates (data not shown), as described previously (43)(44)(45)(46)(47).…”

Section: Discussionmentioning

confidence: 99%

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Alterations of Ceramide/Sphingosine 1-Phosphate Rheostat Involved in the Regulation of Resistance to Imatinib-induced Apoptosis in K562 Human Chronic Myeloid Leukemia Cells

Baran

Salas

Senkal

et al. 2007

Journal of Biological Chemistry

194

195

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In this study, mechanisms of resistance to imatinib-induced apoptosis in human K562 cells were examined. Continuous exposure to stepwise increasing concentrations of imatinib resulted in the selection of K562/IMA-0.2 and -1 cells, which expressed ϳ2.3-and 19-fold resistance, respectively. Measurement of endogenous ceramides by high performance liquid chromatography/mass spectroscopy showed that treatment with imatinib increased the generation of ceramide, mainly C 18 -ceramide, which is generated by the human longevity assurance gene 1 (hLASS1), in sensitive, but not in resistant cells. Inhibition of hLASS1 by small interfering RNA partially prevented imatinib-induced cell death in sensitive cells. In reciprocal experiments, overexpression of hLASS1, and not hLASS6, in drug-resistant cells caused a marked increase in imatinib-induced C 18 -ceramide generation, and enhanced apoptosis. Interestingly, there were no defects in the levels of mRNA and enzyme activity levels of hLASS1 for ceramide generation in K562/IMA-1 cells. However, expression levels of sphingosine kinase-1 (SK1) and generation of sphingosine 1-phosphate (S1P) were increased significantly in K562/IMA-1 cells, channeling sphingoid bases to the sphingosine kinase pathway. The partial inhibition of SK1 expression by small interference RNA modulated S1P levels and increased sensitivity to imatinib-induced apoptosis in resistant cells. On the other hand, forced expression of SK1 in K562 cells increased the ratio between total S1P/C 18 -ceramide levels ϳ6-fold and prevented apoptosis significantly in response to imatinib. Additional data indicated a role for SK1/S1P signaling in the up-regulation of the Bcr-Abl expression at the post-transcriptional level, which suggested a possible mechanism for resistance to imatinib-mediated apoptosis. In conclusion, these data suggest a role for endogenous C 18 -ceramide synthesis mainly via hLASS1 in imatinib-induced apoptosis in sensitive cells, whereas in resistant cells, alterations of the balance between the levels of ceramide and S1P by overexpression of SK1 result in resistance to imatinib-induced apoptosis. Chronic myeloid leukemia (CML)5 is a hematopoietic stem cell disorder (1). There is an elevated but immature white blood cell count in CML. The natural history of CML follows a progression from a chronic phase to an accelerated phase or to a rapidly fatal blast crisis within 3-5 years in patients. Blood cells differentiate normally in the chronic phase but not in the blast phase (2).CML is usually diagnosed by the presence of an abnormal Philadelphia (Ph) chromosome, which results from a translocation between the long arms of chromosomes 9 and 22. This exchange brings together two genes: the Bcr gene on chromosome 22 and the proto-oncogene Abl on chromosome 9 (3). The resulting hybrid gene, Bcr-Abl, encodes for a fusion protein with tyrosine kinase activity, which mediates signal transduction pathways, leading to uncontrolled growth.One of the major advancements in the treatment of CML has been the develo...

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Section: Discussionsupporting

confidence: 58%

Section: Discussionmentioning

confidence: 99%

Alterations of Ceramide/Sphingosine 1-Phosphate Rheostat Involved in the Regulation of Resistance to Imatinib-induced Apoptosis in K562 Human Chronic Myeloid Leukemia Cells

Baran

Salas

Senkal

et al. 2007

Journal of Biological Chemistry

194

195

View full text Add to dashboard Cite

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“…On the basis of confocal microscopy data, this activity was ascribed to mitochondria (24) and plasma membrane (53), and it was also demonstrated in purified mitochondria (25,27 (54). NCDase could also function in the reverse direction, e.g.…”

Section: Discussionmentioning

confidence: 83%

Essential Roles of Neutral Ceramidase and Sphingosine in Mitochondrial Dysfunction Due to Traumatic Brain Injury

Novgorodov

Riley

et al. 2014

Journal of Biological Chemistry

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Background: A cardinal feature of many neurological disorders is mitochondrial dysfunction. Results: Knocking down neutral ceramidase reduces mitochondrial sphingosine, preserves mitochondrial function, and improves brain function recovery after trauma. Conclusion: Activation of the sphingosine-generating pathway plays a significant role in promoting mitochondrial injury. Significance: This is the first direct evidence of endogenous sphingosine involvement in regulation of mitochondrial function.

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“…The pellets were washed twice and then solubilized in 1% (w/v) Triton-X100. Equal amounts of protein from each fraction were subjected to SDS-PAGE and Western blot analysis using antibody specific to the His-tag as described previously (34).…”

Section: Expression Of Pcdasementioning

confidence: 99%

“…The enzymatic reaction was continued for 30 min at 37jC, and it was stopped by addition of chloroform/methanol (1:1). The organic phase was spotted onto TLC plates and analyzed as described previously (34).…”

Section: Cdase Enzyme Assays and Biochemical Characterizationmentioning

confidence: 99%

Large-scale purification and characterization of recombinant Pseudomonas ceramidase: regulation by calcium

Snook

Tani

et al. 2007

Journal of Lipid Research

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Ceramidases (CDases) hydrolyze ceramide to sphingosine (SPH) and fatty acid. Pseudomonas CDase (pCDase) is a homolog of mammalian neutral ceramidases and may play roles in disease pathogenesis. In this study, pCDase was cloned and expressed in Escherichia coli (E. coli). The expressed recombinant pCDase was solubilized by optimizing several factors, including culture medium, the concentration of isopropyl-b-thiogalactopyranoside (IPTG), temperature, and time of induction, which were identified to be critical for the optimal production of recombinant pCDase. The recombinant pCDase was purified using nickel-nitrilotriacetic acid affinity, phenyl-Sepharose, and Q-Sepharose column chromatography, which gave an overall yield of 0.45 mg/l purified protein of starting culture. The activity of the recombinant pCDase followed classical Michaelis-Menten kinetics, with optimum activity in the neutral pH range. Both the hydrolytic and the reverse activities of CDase were stimulated by calcium with an affinity constant (K a ) of 1.5 mM. Kinetics studies showed that calcium caused a decrease of K m and an increase in V max of pCDase. Calcium and D-erythro-sphingosine caused significant changes in the near ultraviolet circular dichroism (CD) spectra and the changes were inhibited in the presence of EGTA. These results identify important interactions between calcium and pCDase, which may play an essential role in the interaction of pCDase and its substrate.-Wu, B. X., C. F. Snook, M. Tani, E. E. Bü llesbach, and Y. A. Hannun. Large-scale purification and characterization of recombinant Pseudomonas ceramidase: regulation by calcium. J. Lipid Res. 2007. 48: 600-608.

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Identification of a novel amidase motif in neutral ceramidase

Cited by 44 publications

References 40 publications

Alterations of Ceramide/Sphingosine 1-Phosphate Rheostat Involved in the Regulation of Resistance to Imatinib-induced Apoptosis in K562 Human Chronic Myeloid Leukemia Cells

Alterations of Ceramide/Sphingosine 1-Phosphate Rheostat Involved in the Regulation of Resistance to Imatinib-induced Apoptosis in K562 Human Chronic Myeloid Leukemia Cells

Essential Roles of Neutral Ceramidase and Sphingosine in Mitochondrial Dysfunction Due to Traumatic Brain Injury

Large-scale purification and characterization of recombinant Pseudomonas ceramidase: regulation by calcium

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