Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 May 7;125(19):2948-57.
doi: 10.1182/blood-2014-07-586511. Epub 2015 Feb 10.

miRs-138 and -424 control palmitoylation-dependent CD95-mediated cell death by targeting acyl protein thioesterases 1 and 2 in CLL

Valeska Berg  1 , Marion Rusch  2 , Nachiket Vartak  3 , Christian Jüngst  4 , Astrid Schauss  4 , Herbert Waldmann  5 , Christian Hedberg  2 , Christian P Pallasch  1 , Philippe I H Bastiaens  6 , Michael Hallek  1 , Clemens-Martin Wendtner  7 , Lukas P Frenzel  1
Affiliations

miRs-138 and -424 control palmitoylation-dependent CD95-mediated cell death by targeting acyl protein thioesterases 1 and 2 in CLL

Valeska Berg et al. Blood. .

Abstract

Resistance toward CD95-mediated apoptosis is a hallmark of many different malignancies, as it is known from primary chronic lymphocytic leukemia (CLL) cells. Previously, we could show that miR-138 and -424 are downregulated in CLL cells. Here, we identified 2 new target genes, namely acyl protein thioesterase (APT) 1 and 2, which are under control of both miRs and thereby significantly overexpressed in CLL cells. APTs are the only enzymes known to promote depalmitoylation. Indeed, membrane proteins are significantly less palmitoylated in CLL cells compared with normal B cells. We identified APTs to directly interact with CD95 to promote depalmitoylation, thus impairing apoptosis mediated through CD95. Specific inhibition of APTs by siRNAs, treatment with miRs-138/-424, and pharmacologic approaches restore CD95-mediated apoptosis in CLL cells and other cancer cells, pointing to an important regulatory role of APTs in CD95 apoptosis. The identification of the depalmitoylation reaction of CD95 by APTs as a microRNA (miRNA) target provides a novel molecular mechanism for how malignant cells escape from CD95-mediated apoptosis. Here, we introduce palmitoylation as a novel posttranslational modification in CLL, which might impact on localization, mobility, and function of molecules, survival signaling, and migration.

PubMed Disclaimer

Figures

Figure 1
Figure 1
APT1 and APT2 are regulated by miR-138 and miR-424. Quantitative PCR was used to compare expression of miRNAs potentially regulating APT1 and APT2 in CLL cells (n = 19 [miR-138] or n = 7 [miR-424]) and healthy B cells (n = 9 [miR-138] or n = 5 [miR-424]). miR-138 and miR-424 are significantly downregulated in primary CLL cells (A). Luciferase assays were performed to analyze the binding of both miRNAs to the respective 3′-UTR of APT1 or APT2. It could be shown that both miRNAs are in principle able to regulate APT1 (n = 4) or APT2 expression (n = 3) (B). By western blotting it could be demonstrated that miR-138 regulates APT1 (n = 3) and miR-424 regulates APT2 protein expression (n = 3) (C). MiR-138 and miR-424 regulate APT1- and APT2-mRNA levels by reducing mRNA levels (D). Means are given with their SEM. The statistical significance was determined using the Student t test. *P < .05, **P < .01, or ***P < .001 were considered significant. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SEM, standard error of the mean.
Figure 2
Figure 2
APT1 and APT2 are overexpressed in primary CLL cells ex vivo. APT1 and APT2 mRNA expression was analyzed in healthy B cells (n = 4) and CLL cells (n = 7 [APT1] or n = 8 [APT2]) by quantitative PCR (A). Western blotting showed that APT1 and APT2 are overexpressed not only on mRNA, but also on protein level in CLL cells (n = 7 [APT1] or n = 9 [APT2]) compared with healthy B cells (n = 4 [APT1] or n = 5 [APT2]) (B-C). Global palmitoylation was analyzed by click chemistry. Comparison of CLL cells (n = 10), healthy PBMCs (n = 6), and healthy B cells (n = 6) revealed that the global palmitoylation level is significantly lower in CLL cells than in the healthy controls (D). Palmitoylation of proteins can be enhanced by the use of the depalmitoylation inhibitor PB (n = 5) (E). Means are given with their SEM. The statistical significance was determined using the Student t test. *P < .05, **P < .01, or ***P < .001 were considered significant.
Figure 3
Figure 3
CD95 is depalmitoylated by APTs. Principle of ABE assays according to Wan et al (A). Palmitoylation of CD95 was analyzed by ABE assays. Therefore, CD95 of primary CLL cells was immunoprecipitated. Inhibition of depalmitoylation by PB significantly increased palmitoylation levels of CD95 (n = 3) (B).
Figure 4
Figure 4
CD95 and APTs interact at the plasma membrane. FLIM-FRET experiments of CD95 with APT1 and APT2 demonstrate that CD95 directly interacts with both proteins (A). The interaction is dependent on the palmitoylation of cysteine 199 (B). Lifetime of the donor (CD95-eYFP) at the plasma membrane and in the cytoplasm is significantly reduced after incubation with the acceptor (APT1- or APT2-mCherry) (C). Means are given with their SEM. The statistical significance was determined using the Student t test. *P < .05, **P < .01, or ***P < .001 were considered significant.
Figure 5
Figure 5
Mobility of CD95 within the plasma membrane is regulated by palmitoylation. FRAP assays were performed to investigate the impact of palmitoylation on the receptor’s mobility on the plasma membrane. A typical experiment is shown for CD95-eYFP and CD95-eYFP mut (A). Comparison of wild-type (palmitoylated) CD95 with mutated (C199S, unpalmitoylated) CD95 showed that the mobile fraction is significantly higher in cells expressing the unpalmitoylated receptor (n = 15), while t1/2 is unaffected (B-C). Means are given with their SEM. The statistical significance was determined using the Student t test. *P < .05, **P < .01, or ***P < .001 were considered significant.
Figure 6
Figure 6
APT1 and APT2 control CD95-mediated apoptosis. PB treatment significantly enhanced apoptosis mediated through anti-CD95 (CH11) in CLL cells after 24 hours (n = 14). Apoptosis of CLL cells was determined by Annexin V/7-AAD staining and subsequent flow cytometry. Apoptosis was also significantly increased after treatment of HeLa cells with PB and CD95 after 24 hours (n = 3). Survival of HeLa cells was determined by XTT assays (A). Specific siRNA knockdown of APT1 (n = 3) or APT2 (n = 5) for 48 hours was able to mimic the PB effect in CLL cells (B) and in HeLa cells (n = 3) (C). Knockdown efficiency was determined by western blotting (B-C). In addition, overexpression of miRNAs regulating APT1 and APT2 for 48 hours also led to increased apoptosis in HeLa cells (n = 4) (D). Means are given with their SEM. The statistical significance was determined using the Student t test. *P < .05, **P < .01, or ***P < .001 were considered significant.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. de Totero D, Montera M, Rosso O, et al. Resistance to CD95-mediated apoptosis of CD40-activated chronic lymphocytic leukemia B cells is not related to lack of DISC molecules expression. Hematol J. 2004;5(2):152–160. - PubMed
    1. Kater AP, Dicker F, Mangiola M, et al. Inhibitors of XIAP sensitize CD40-activated chronic lymphocytic leukemia cells to CD95-mediated apoptosis. Blood. 2005;106(5):1742–1748. - PMC - PubMed
    1. Romano C, De Fanis U, Sellitto A, et al. Induction of CD95 upregulation does not render chronic lymphocytic leukemia B-cells susceptible to CD95-mediated apoptosis. Immunol Lett. 2005;97(1):131–139. - PubMed
    1. Dicker F, Kater AP, Fukuda T, Kipps TJ. Fas-ligand (CD178) and TRAIL synergistically induce apoptosis of CD40-activated chronic lymphocytic leukemia B cells. Blood. 2005;105(8):3193–3198. - PubMed
    1. Krammer PH. CD95’s deadly mission in the immune system. Nature. 2000;407(6805):789–795. - PubMed

Publication types

MeSH terms