Ceramide binds to the CaLB domain of cytosolic phospholipase A2 and facilitates its membrane docking and arachidonic acid release
Ceramide binds to the CaLB domain of cytosolic phospholipase A2 and facilitates its membrane docking and arachidonic acid release
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神经酰胺与胞质磷脂酶 A2 的 CaLB 结构域结合，促进其膜对接和花生四烯酸释放
DOI：
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
J. Pfeilschifter
中科院分区：
文献类型：
--
作者：
A. Huwiler;B. Johansen;Anita Skarstad;J. Pfeilschifter
类二十烷酸的过量产生是许多炎性疾病的特征。在这项研究中，我们发现神经酰胺是炎症细胞因子作用的早期信使，对细胞溶质磷脂酶A2（cPLA 2），花生四烯酸释放和随后类花生酸形成的限速酶产生双重作用。用外源性短链神经酰胺类似物刺激肾系膜细胞30和60分钟，导致花生四烯酸释放的浓度依赖性增加，不被丝裂原活化蛋白激酶途径的特异性抑制剂阻断。这表明这些已建立的cPLA 2上游激活剂不参与神经酰胺诱导的花生四烯酸释放。通过使用可光活化的神经酰胺类似物，D-和L-[125 I]3-三氟甲基-3-（间碘苯基）二氮杂环丙烯神经酰胺（TID-神经酰胺），我们观察到神经酰胺与cPLA 2的直接相互作用。这种相互作用与绝对构型无关，因为D-和L-TID-神经酰胺在与cPLA 2结合方面同样有效。此外，cPLA 2的重组CaLB结构域以及cPLA 2的连接“铰链”结构域缺陷的突变体有效地结合D-和L-TID-神经酰胺，而催化结构域不与TID-神经酰胺相互作用。体外结合试验表明，含有增加mol%神经酰胺的硬脂酰-花生四烯酰-磷脂酰胆碱（SAPC）-脂质体导致重组cPLA 2与脂质体的缔合增加。此外，体外cPLA 2活性的测量表明，SAPC-脂质体的存在仅导致弱cPLA 2活性。然而，通过向脂质体中添加神经酰胺，活性显著增加。此外，含有SAPC和鞘磷脂的脂质体没有产生比SAPC脂质体更好的底物，除非加入细菌鞘磷脂酶以产生神经酰胺，然后引起cPLA 2活性的显著增加。这些结果表明，神经酰胺可以通过CaLB结构域直接与cPLA 2相互作用，从而作为膜对接装置，促进cPLA 2在炎症性疾病中的作用。
Excessive production of eicosanoids is characteristic of many inflammatory diseases. In this study we show that ceramide, which is an early messenger of inflammatory cytokine action, exerts a dual effect on the cytosolic phospholipase A2 (cPLA2), the rate‐limiting enzyme in arachidonic acid release and subsequent eicosanoid formation. Stimulation of renal mesangial cells with exogenous short‐chain ceramide analogs for 30 and 60 min leads to a concentration‐dependent increase in arachidonic acid release that is not blocked by specific inhibitors of mitogen‐activated protein kinase pathways. This suggests that these established upstream activators of cPLA2 are not involved in ceramide‐induced arachidonic acid release. By use of photoactivatable ceramide analogs, D‐ and L‐ [125I]3‐trifluoromethyl‐3‐(m‐iodophenyl)diazirineceramides (TID‐ceramides), we observed a direct interaction of ceramide with cPLA2. This interaction was independent of the absolute configuration as D‐ and L‐TID‐ceramide were equally effective in binding to cPLA2. Moreover, recombinant CaLB domain of cPLA2 as well as a mutant deficient in the connecting ‘hinge’ domain of cPLA2, efficiently bound D‐ and L‐TID‐ceramides, whereas the catalytic domain did not interact with TID‐ceramides. In vitro binding assays reveal that stearoyl‐arachidonyl‐phosphatidylcholine (SAPC)‐liposomes containing increasing mol% of ceramide lead to an increased association of recombinant cPLA2 to the liposomes. Furthermore, measurement of cPLA2 activity in vitro shows that the presence of SAPC‐liposomes resulted in only weak cPLA2 activity. However, the activity dramatically increases by addition of ceramide to the liposomes. Furthermore, liposomes containing SAPC and sphingomyelin resulted in no better substrate than SAPC liposomes, unless bacterial sphingomyelinase was added to generate ceramide, which then causes a marked increase in cPLA2 activity. These results demonstrate that ceramide can interact directly with cPLA2 via the CaLB domain and thereby serves as a membrane‐docking device that facilitates cPLA2 action in inflammatory diseases.
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DOI：
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发表时间：
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期刊：
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DOI：
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1990
期刊：
Biochimica et biophysica acta
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