ADA2 is a lysosomal DNase regulating the type-I interferon response

Boehler, Vincent; Bartok, Eva; Krausz, Máté; Polyzou, Aikaterini; Schepp, Johanna; Seidl, Maximilian; Olsen, Jan Ole; Smulski, Cristian R.; Raieli, Salvatore; Hübscher, Katrin; Trompouki, Eirini; Link, Regina; Ebersbach, Hilmar; Honnappa, Srinivas; Marchant, Martine; Staab, Dieter; Guerini, Danilo; Baasch, Sebastian; Henneke, Philipp; Kochs, Georg; Hartmann, Gunther; Geiger, Roger; Grimbacher, Bodo; Warncke, Max; Proietti, Michele

doi:10.1101/2020.06.21.162990

Cited by 10 publications

(9 citation statements)

References 46 publications

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“…LMW-ADA2 protein levels did however not increase upon proteasome inhibition ( Figure S8C ). We therefore assume that glycan trimming and retro-translocation of LMW-ADA2 generate a functional cytosolic or lysosomal protein that might be different in function of secreted HMW-ADA2, as suggested by Greiner-Tollersrud and colleagues (17).…”

Section: Discussionmentioning

confidence: 90%

“…Differences in the molecular weight of a protein can be caused by posttranslational protein modifications and N-glycosylation of ADA2 has previously been confirmed (10,12,17). Therefore, we initially hypothesized that the LMW-form of ADA2 unique to cells expressing WT ADA2 corresponded to glycan-free ADA2.…”

Section: Human Monocyte-derived Macrophages Intracellularly Express A...mentioning

confidence: 97%

“…Research into alternative functions of the ADA2 protein and DADA2-causing variants is therefore warranted. Biochemical characterization of pathogenic ADA2 variants has until now only been performed in a HEK293T overexpression system with supraphysiological intracellular levels of ADA2 while expression of the mutant protein has been shown to be minimal in DADA2 CD14+ monocytes (Chen et al, 2023;Greiner-Tollersrud et al, 2020). Disease-associated ADA2 variants have to date hardly been studied in their physiological milieu.…”

Section: Introductionmentioning

confidence: 99%

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Human ADA2 deficiency is characterized by the absence of an intracellular hypoglycosylated form of adenosine deaminase 2

Ehlers,

Hombrouck,

Wouters

et al. 2023

Preprint

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Pathogenic variants inADA2underlie human deficiency of adenosine deaminase 2 (DADA2) and lead to impaired ADA2 protein secretion and reduced deaminase activity. Yet, the mechanisms driving the disease on a cellular level are poorly understood. Here, we report thatin vitrodifferentiation of macrophages from DADA2 CD14+ monocytes partially restores protein expression of mutant ADA2. Healthy control macrophages express a high-molecular-weight (HMW) secreted form of ADA2 and a hypoglycosylated low-molecular-weight (LMW) intracellular protein. LMW-ADA2 is absent in DADA2 macrophages, suggesting differential glycosylation of mutant ADA2. In HEK293T cells transfected with pathogenic ADA2 variants, HMW-ADA2 accumulates intracellularly. Interactome analysis of the pathogenic variant p.R169Q revealed increased binding to proteins of the protein folding machinery compared with wild-type ADA2. Pathogenic ADA2 variants form intracellular protein aggregates and show increased degradation upon inhibition of the secretory pathway. In conclusion, we show aberrant N-glycosylation of ADA2 expressed in DADA2 macrophages for the first time and establish the absence of LMW-ADA2 as a characteristic of these ADA2-deficient cells.Graphical abstractFigure 1:SummaryThis study identifies a previously undescribed hypoglycosylated intracellular form of ADA2 in human monocyte-derived macrophages. Absence of this low-molecular-weight form is a key feature of human ADA2 deficiency (DADA2), suggesting a role for intracellular ADA2 in the pathogenesis of the disease.

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Section: Discussionmentioning

confidence: 90%

Section: Human Monocyte-derived Macrophages Intracellularly Express A...mentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Human ADA2 deficiency is characterized by the absence of an intracellular hypoglycosylated form of adenosine deaminase 2

Ehlers,

Hombrouck,

Wouters

et al. 2023

Preprint

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“…Therefore, it is likely that ADA2 plays a very insignificant role, if any at all, in the metabolism of adenosine in beta-cells. Instead, it has recently been reported that ADA2 functions as a growth factor ( 39 ) or possibly as an interferon-I response factor ( 40 ), which may explain why ADA2 overexpression instead potentiates beta-cell death in the presence of a high adenosine concentration.…”

Section: Discussionmentioning

confidence: 99%

“…Intracellular adenosine levels were analysed using an ELISA and expressed per mg protein. Frontiers in Endocrinology frontiersin.org it has recently been reported that ADA2 functions as a growth factor (39) or possibly as an interferon-I response factor (40), which may explain why ADA2 overexpression instead potentiates beta-cell death in the presence of a high adenosine concentration. The low expression level of the ADA1 protein in EndoC-betaH1 cells is probably explained, at least in part, by miRNA-mediated suppression of ADA1 translation.…”

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confidence: 99%

Metabolic stress-induced human beta-cell death is mediated by increased intracellular levels of adenosine

et al. 2023

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IntroductionHigh intracellular concentrations of adenosine and 2’-deoxyadenosine have been suggested to be an important mediator of cell death. The aim of the present study was to characterize adenosine-induced death in insulin-producing beta-cells, at control and high glucose + palmitate-induced stress conditions.MethodsHuman insulin-producing EndoC-betaH1 cells were treated with adenosine, 2’-deoxyadenosine, inosine and high glucose + sodium palmitate, and death rates using flow cytometry were studied.ResultsWe observed that adenosine and the non-receptor-activating analogue 2-deoxyadenosine, but not the adenosine deamination product inosine, promoted beta-cell apoptosis at concentrations exceeding maximal adenosine-receptor stimulating concentrations. Both adenosine and inosine were efficiently taken up by EndoC-betaH1 cells, and inosine counteracted the cell death promoting effect of adenosine by competing with adenosine for uptake. Both adenosine and 2’-deoxyadenosine promptly reduced insulin-stimulated production of plasma membrane PI(3,4,5)P3, an effect that was reversed upon wash out of adenosine. In line with this, adenosine, but not inosine, rapidly diminished Akt phosphorylation. Both pharmacological Bax inhibition and Akt activation blocked adenosine-induced beta-cell apoptosis, indicating that adenosine/2’-deoxyadenosine inhibits the PI3K/Akt/BAD anti-apoptotic pathway. High glucose + palmitate-induced cell death was paralleled by increased intracellular adenosine and inosine levels. Overexpression of adenosine deaminase-1 (ADA1) in EndoC-betaH1 cells, which increased Akt phosphorylation, prevented both adenosine-induced apoptosis and high glucose + palmitate-induced necrosis. ADA2 overexpression not only failed to protect against adenosine and high glucose + palmitate-activated cell death, but instead potentiated the apoptosis-stimulating effect of adenosine. In line with this, ADA1 overexpression increased inosine production from adenosine-exposed cells, whereas ADA2 did not. Knockdown of ADA1 resulted in increased cell death rates in response to both adenosine and high glucose + palmitate. Inhibition of miR-30e-3p binding to the ADA1 mRNA 3’-UTR promoted the opposite effects on cell death rates and reduced intracellular adenosine contents.DiscussionIt is concluded that intracellular adenosine/2’-deoxyadenosine regulates negatively the PI3K pathway and is therefore an important mediator of beta-cell apoptosis. Adenosine levels are controlled, at least in part, by ADA1, and strategies to upregulate ADA1 activity, during conditions of metabolic stress, could be useful in attempts to preserve beta-cell mass in diabetes.

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Mechanisms of vascular inflammation in deficiency of adenosine deaminase 2 (DADA2)

2022

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ADA2 is a lysosomal DNase regulating the type-I interferon response

Cited by 10 publications

References 46 publications

Human ADA2 deficiency is characterized by the absence of an intracellular hypoglycosylated form of adenosine deaminase 2

Human ADA2 deficiency is characterized by the absence of an intracellular hypoglycosylated form of adenosine deaminase 2

Metabolic stress-induced human beta-cell death is mediated by increased intracellular levels of adenosine

Mechanisms of vascular inflammation in deficiency of adenosine deaminase 2 (DADA2)

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